Cross-reactive antibodies against dengue virus and uses thereof

ABSTRACT

The invention relates to a human anti-dengue virus antibody (an anti-DENV antibody) that binds to a DENV envelope protein and is cross-reactive with DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4. The disclosure provides an anti-DENV antibody that cross-reacts with and neutralizes all four DENV serotypes. Also provided is a nucleic acid molecule that encodes such an anti-DENV antibody. Also provided is a method to produce and use such an antibody or nucleic acid molecule encoding such an antibody.

FIELD

The invention relates to an antibody against Dengue virus. Such antibodyis a human antibody that cross-reacts with all four serotypes of Denguevirus.

BACKGROUND/INTRODUCTION Dengue virus (DENV) causes the most prevalentmosquito-borne viral disease: over 2.5 billion people are at risk forinfection in over 100 countries. Each year, dengue virus infects andexhibits symptoms in about 100 million people worldwide. Of thoseinfected, about 250,000-500,000 develop severe illness, and up to 50,000die from dengue hemorrhagic fever (DHF) each year (see, e.g., Dengue anddengue hemorrhagic fever, World Health Organization Media center, FactSheet No. 117, March 2009; Deen J L et al., 2006, Lancet 368, 170-173;Kyle J L et al., 2008, Annu Rev Microbiol 62, 71-92). In 2010, denguevirus re-entered the US via the Florida Keys, after an absence ofsixty-five years (see, e.g., Homeland Security News Wire, 2 Jun. 2010).

There are four closely related, but antigenically distinct, dengue virusserotypes, namely DENV serotype 1 (DENV-1), DENV serotype 2 (DENV-2),DENV serotype 3 (DENV-3), and DENV serotype 4 (DENV-4). Each serotypecomprises several genotypes that exhibit differences in their infectioncharacteristics in both the mosquito vector and the human host. Recoveryfrom infection by one serotype provides lifelong immunity against thatserotype, but confers only partial and transient protection againstsubsequent infection by other serotypes. One of the confounding problemsthat has faced vaccine development for decades has been this inabilityof one serotype to protect against infection by another. Rather, theinduced humoral immune response to one DENV serotype as a result ofinfection can enhance the infection and disease processes brought aboutby a subsequent infection with another DENV serotype. Such subsequentinfection is thought to increase the risk of developing denguehemorrhagic fever (DHV), a potentially lethal condition. (see, e.g., WHOFact Sheet No. 117, ibid.; Halstead S B et al., 2007, Lancet 370,1644-1652; Dejnirattisai W, et al., 2010, Science 328, 745-748.)

Dengue virus is a single positive-stranded RNA virus of theFlaviviridae, genus Flavivirus. The viral genome of about 10.7 kilobasesis translated as a single polypeptide that is cleaved into threestructural proteins (namely a capsid protein (C), a membrane protein (M,prM/M), and an envelope protein (E)), and seven non-structural proteins(namely NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).

The initial step of dengue virus infection is entry of the virus intocells; such entry is mediated by DENV envelope E, a glycoprotein (see,e.g., Dimitrov D S, 2004, Nat. Rev. Microbiol. 2, 109-122). DENV E is atype II fusion protein and consists of three domains (see, e.g.,Dimitrov D S, ibid.; Kuhn R J et al., 2002, Cell 108, 717-725). Thedomains are named ED1, ED2, and ED3, respectively; other names for theenvelope domains are domain I (DI), domain II (DII), and domain III(DIII, or Env-DIII), respectively. ED3 has been proposed to contain areceptor binding domain (Huerta V et al., 2008, Virus Res 137, 225-234;Crill W D et al., 2001, J Virol 75, 7769-7773).

Antibodies are important for protective and pathogenic immune responsesto dengue virus (see, e.g., Halstead et al., ibid.). Their major targetis the DENV E protein (see, e.g., Marasco W A et al., 2007, NatBiotechnol 25, 1421-1434; Modis Y et al., 2005, J Virol 79, 1223-1231;Roehrig J T et al., 1998, Virology 246, 317-328; Bedouelle H et al.,2006, FEBS J 273, 34-46; Thullier P et al., 2001, J Gen Virol 82,1885-1892). The amino acid sequences of E proteins for DENV-1, DENV-2,DENV-3, and DENV-4 differ by 25% to 40%; the amino acid sequences of Eproteins for genotypes within a DENV serotype vary by up to 3% (see,e.g., Holmes E C et al., 2003, Infect Genet Evol 3, 19-28). Antibodiesagainst ED3 are both serotype specific and cross-reactive; suchantibodies are effective DENV neutralizers, typically better thanantibodies against ED1 or ED2 (see, e.g., de Alwis R, et al., 2011, PLoSNegl Trop Dis 5, e1188; Balsitis S J, et al., 2010, PLoS Pathog 6,e1000790; Beltramello M et al., 2010, Cell Host Microbe 8, 271-283;Sukupolvi-Petty S et al., 2010, J Virol 84, 9227-9239; Shrestha B etal., 2010, PLoS Pathog 6, e1000823; Rajamanonmani R et al., 2009, J GenVirol 90, 799-809; Sukupolvi-Petty S et al., 2007, J Virol 81,12816-12826; Gromowski G D et al., 2010, Virology 407, 237-246; Matsui Ket al., 2010, J Gen Virol 91, 2249-2253; Matsui K et al., 2009, Virology384, 16-20; Gromowski G D et al., 2008, J Virol 82, 8828-8837; GromowskiGD et al., 2007, Virology 366, 349-360; Hiramatsu K et al., 1996,Virology 224, 437-445; Lok S M et al., 2008, Nat Struct Mol Biol 15,312-317). The cross-reactive antibodies are generally weakerneutralizers than the serotype-specific antibodies (see, e.g.,Sukupolvi-Petty S et al, 2007, ibid.; Gromowski G D et al., 2008,ibid.). Anti-ED3 serotype-specific and cross-reactive antibodies areelicited using ED3 as a vaccine immunogen (see, e.g., Guzman M G et al.,2010, Vaccines 9, 137-147; Bernardo L et al., 2011, Vaccine 29,4256-4263; Bernardo L et al., 2009, Clin Vaccine Immunol 16, 1829-1831;Izquierdo A et al., 2008, Virus Res 138, 135-138; Simmons M et al.,1998, Am J Trop Med Hyg 58, 655-662; Simmons M et al., 2001, Am J TropMed Hyg 65, 159-161; Srivastava A K et al., 1995, Vaccine 13, 1251-1258)and in infected humans (see, e.g., de Alwis R et al., ibid.; Midgley CM, 2011, J. Virol. 85, 410-421; Rothman A L, 2004, J. Clin. Invest. 113,946-951; Wahala, W M et al., 2009, Virology 392, 103-113). Recently,several human monoclonal antibodies were selected from immortalized Bcells obtained from DENV-infected people, a few of which also exhibitedsome cross-reactive neutralizing activity against all four serotypes; acocktail of three of antibodies targeting distinct epitopes andengineered to prevent FcγR binding was successful in a mouse model ofdisease when administered one day after challenge (Beltramello M et al,2010, ibid.).

There remains a need for an effective therapeutic, an effectiveprophylactic, or an effective therapeutic and prophylactic againstdengue virus infection, not only to protect an individual from aninitial infection, but also to protect that individual from subsequentinfection not only by the same DENV serotype but also by any and allother DENV serotypes. As such, there is a need for an anti-DENV antibodythat cross-reacts with and neutralizes DENV-1, DENV-2, DENV-3, andDENV-4 dengue virus serotypes. There is also a need for a vaccine thatcomprises an epitope recognized by such a cross-reactive anti-DENVantibody to protect an individual from dengue virus infection.

SUMMARY

The present disclosure provides a human anti-dengue virus antibody (ananti-DENV antibody) that (a) binds to a DENV envelope protein and (b) iscross-reactive with DENV serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4. In one embodiment, such an anti-DENV antibodyneutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4. Also provided is a nucleic acid molecule that encodes suchan anti-DENV antibody. Also provided is a method to produce and use suchan antibody or a nucleic acid molecule encoding such an antibody. Thepresent disclosure further provides a protein comprising an epitope thatbinds to an anti-DENV antibody that (a) binds to a DENV envelope proteinand (b) cross-reacts with DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4. Also provided is a nucleic acidmolecule that encodes such a protein. Also provided is a method toproduce and use such a protein or a nucleic acid molecule encoding sucha protein.

The disclosure provides a human anti-dengue virus antibody that binds todomain III of an envelope protein of dengue virus, wherein the antibodyis cross-reactive with domain III of dengue virus (DENV) serotype 1envelope protein, domain III of DENV serotype 2 envelope protein, domainIII of DENV serotype 3 envelope protein, and domain III of DENV serotype4 envelope protein. In some embodiments the antibody does not enhanceDengue virus infection.

The disclosure also provides a human anti-dengue virus bispecificantibody that comprises two antibodies each of which binds to domain IIIof an envelope protein of dengue virus and at least one of which iscross-reactive with domain III of dengue virus (DENV) serotype 1envelope protein, domain III of DENV serotype 2 envelope protein, domainIII of DENV serotype 3 envelope protein, and domain III of DENV serotype4 envelope protein. In some embodiments, both antibodies arecross-reactive with domain III of dengue virus (DENV) serotype 1envelope protein, domain III of DENV serotype 2 envelope protein, domainIII of DENV serotype 3 envelope protein, and domain III of DENV serotype4 envelope protein. In some embodiments, the bispecific antibody doesnot enhance Dengue virus infection.

The disclosure also provides a human anti-dengue virus antibody thatbinds to domain III of an envelope protein of dengue virus, wherein theantibody is cross-reactive with domain III of dengue virus (DENV)serotype 1 envelope protein, domain III of DENV serotype 2 envelopeprotein, domain III of DENV serotype 3 envelope protein, and domain IIIof DENV serotype 4 envelope protein, and wherein the antibody isselected from the group consisting of: an antibody comprising acomplementarity determining region (CDR) having an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,and combinations thereof; an antibody comprising a complementaritydetermining region (CDR) having an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinationsthereof; and an antibody comprising a complementarity determining region(CDR) having an amino acid sequence selected from the group consistingof amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations thereof.

The disclosure also provides a human anti-dengue virus antibody thatbinds to domain III of an envelope protein of dengue virus, wherein theantibody is cross-reactive with domain III of dengue virus (DENV)serotype 1 envelope protein, domain III of DENV serotype 2 envelopeprotein, domain III of DENV serotype 3 envelope protein, and domain IIIof DENV serotype 4 envelope protein, and wherein the antibody comprisesa CDR-H3 having an amino acid sequence selected from the groupconsisting of SEQ ID NO:12, SEQ ID NO:32, and SEQ ID NO:52.

In some embodiments, the antibody neutralizes DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4.

In some embodiments, the antibody binds to each of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4 envelope proteins witha dissociation constant (K_(D)) of no more than about 40 nanomolar (nM).

In some embodiments, such an antibody binds to each of DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope proteinswith a K_(D) of no more than about 20 nM.

In some embodiments, such an antibody binds to each of DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope proteinswith a K_(D) of no more than about 1 nM.

In some embodiments, such an antibody binds to each of three DENVserotypes with a K_(D) of no more than about 1 nM and binds to thefourth DENV serotype with a K_(D) of no more than about 40 nM.

In some embodiments, such an antibody neutralizes each of DENV serotype1, DENV serotype 2, DENV serotype 3, and DENV serotype 4 at an IC₅₀ ofless than about 25 micrograms per ml (μg/ml).

In some embodiments, such an antibody neutralizes each of DENV serotype1, DENV serotype 2, DENV serotype 3, and DENV serotype 4 at an IC₅₀ ofless than about 1 μg/ml.

In some embodiments, such an antibody binds to each of DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope proteinswith a K_(D) of no more than about 1 nM, and wherein the antibodyneutralizes each of DENV serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4 at an IC₅₀ of less than about 1 μg/ml.

In some embodiments, such an antibody is of an isotype selected from thegroup consisting of IgG, IgM, and IgA.

In some embodiments, such an antibody is not isolated from a humansubject.

In some embodiments, such an antibody is selected from the groupconsisting of a full length Ig antibody, a full-length soluble antibody,a monospecific antibody, a bispecific antibody, a multi-specificantibody, a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, asingle domain heavy chain antibody, a single domain light chainantibody, and a complementarity determining region (CDR).

In some embodiments, such an antibody is selected from the groupconsisting of: an antibody comprising an amino acid sequence that is atleast about 90 percent identical to an amino acid sequence selected fromthe group consisting of amino acid sequences SEQ ID NO:4, SEQ ID NO:6,and SEQ ID NO:2; an antibody comprising an amino acid sequence that isat least about 90 percent identical to an amino acid sequence selectedfrom the group consisting of amino acid sequences SEQ ID NO:24, SEQ IDNO:26, and SEQ ID NO:22; and an antibody comprising an amino acidsequence that is at least about 90 percent identical to an amino acidsequence selected from the group consisting of amino acid sequences SEQID NO:44, SEQ ID NO:46, and SEQ ID NO:42.

In some embodiments, such an antibody is selected from the groupconsisting of: an antibody comprising an amino acid sequence selectedfrom the group consisting of amino acid sequences SEQ ID NO:4, SEQ IDNO:6, and SEQ ID NO:2; an antibody comprising an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:24,SEQ ID NO:26, and SEQ ID NO:22; and an antibody comprising an amino acidsequence selected from the group consisting of amino acid sequences SEQID NO:44, SEQ ID NO:46, and SEQ ID NO:42.

In some embodiments, such an antibody comprises a V_(H) chain comprisingamino acid sequence SEQ ID NO:4 and a V_(L) chain comprising amino acidsequence SEQ ID NO:6.

In some embodiments, such an antibody comprises a V_(H) chain comprisingamino acid sequence SEQ ID NO:24 and a V_(L) chain comprising amino acidsequence SEQ ID NO:26.

In some embodiments, such an antibody comprises a V_(H) chain comprisingamino acid sequence SEQ ID NO:44 and a V_(L) chain comprising amino acidsequence SEQ ID NO:46.

In some embodiments, such an antibody is selected from the groupconsisting of: an antibody comprising a first amino acid sequence thatis at least about 90 percent identical to amino acid sequence SEQ IDNO:4 and a second amino acid sequence that is at least about 90 percentidentical to amino acid sequence SEQ ID NO:6, wherein the first aminoacid sequence and second amino acid sequence are joined by a peptidelinker; an antibody comprising a first amino acid sequence that is atleast about 90 percent identical to amino acid sequence SEQ ID NO:24 anda second amino acid sequence that is at least about 90 percent identicalto amino acid sequence SEQ ID NO:26, wherein the first amino acidsequence and second amino acid sequence are joined by a peptide linker;and an antibody comprising a first amino acid sequence that is at leastabout 90 percent identical to amino acid sequence SEQ ID NO:44 and asecond amino acid sequence that is at least about 90 percent identicalto amino acid sequence SEQ ID NO:46, wherein the first amino acidsequence and second amino acid sequence are joined by a peptide linker.

In some embodiments, such an antibody is selected from the groupconsisting of: an antibody comprising amino acid sequence SEQ ID NO:4and amino acid sequence SEQ ID NO:6, wherein amino acid sequence SEQ IDNO:4 and amino acid sequence SEQ ID NO:6 are joined by a peptide linker;an antibody comprising amino acid sequence SEQ ID NO:24 and amino acidsequence SEQ ID NO:26, wherein amino acid sequence SEQ ID NO:24 andamino acid sequence SEQ ID NO:26 are joined by a peptide linker; and anantibody comprising amino acid sequence SEQ ID NO:44 and amino acidsequence SEQ ID NO:46, wherein amino acid sequence SEQ ID NO:44 andamino acid sequence SEQ ID NO:46 are joined by a peptide linker.

In some embodiments, the linker is about 10 to about 25 amino acids inlength.

In some embodiments, the linker comprises an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:20,SEQ ID NO:40, and SEQ ID NO:60.

In some embodiments, an antibody of the disclosure comprises at leastone complementarity determining region (CDR) of an antibody selectedfrom the group consisting of human anti-dengue virus antibody m366,human anti-dengue virus antibody m366.6, and human anti-dengue virusantibody m360.6.

In some embodiments, such an antibody is selected from the groupconsisting of: an antibody comprising a CDR having an amino acidsequence selected from the group consisting of amino acid sequences SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, and combinations thereof; an antibody comprising acomplementarity determining region (CDR) having an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 andcombinations thereof; and an antibody comprising a complementaritydetermining region (CDR) having an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and combinationsthereof.

In some embodiments, such an antibody is selected from the groupconsisting of: an antibody comprising a CDR-H1 having amino acidsequence SEQ ID NO:8, a CDR-H2 having amino acid sequence SEQ ID NO:10,a CDR-H3 having amino acid sequence SEQ ID NO:12, a CDR-L1 having aminoacid sequence SEQ ID NO:14, a CDR-L2 having amino acid sequence SEQ IDNO:16, and a CDR-L3 having amino acid sequence SEQ ID NO:18; an antibodycomprising a CDR-H1 having amino acid sequence SEQ ID NO:28, a CDR-H2having amino acid sequence SEQ ID NO:30, a CDR-H3 having amino acidsequence SEQ ID NO:32, a CDR-L1 having amino acid sequence SEQ ID NO:34,a CDR-L2 having amino acid sequence SEQ ID NO:36, and a CDR-L3 havingamino acid sequence SEQ ID NO:38; and an antibody comprising a CDR-H1having amino acid sequence SEQ ID NO:48, a CDR-H2 having amino acidsequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ ID NO:52,a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 having aminoacid sequence SEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQID NO:58.

In some embodiments, such an antibody comprises an amino acid sequencethat is at least about 90 percent identical to an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:2,SEQ ID NO:22, and SEQ ID NO:42.

In some embodiments, such an antibody comprises amino acid sequence SEQID NO:2.

In some embodiments, such an antibody comprises amino acid sequence SEQID NO:22.

In some embodiments, such an antibody comprises amino acid sequence SEQID NO:42.

In some embodiments, such an antibody comprises an Fc domain.

In some embodiments, such an antibody is a bispecific antibody.

In some embodiments, such a bispecific antibody comprises an antibody ofany of the embodiments of the disclosure.

In some embodiments, such a bispecific antibody comprises at least oneantibody comprises a CDR-H3 having an amino acid sequence selected fromthe group consisting of SEQ ID NO:12, SEQ ID NO:32, and SEQ ID NO:52. Insome embodiments, both antibodies of such a bispecific antibody comprisea CDR-H3 having an amino acid sequence selected from the groupconsisting of SEQ ID NO:12, SEQ ID NO:32, and SEQ ID NO:52.

In some embodiments, such a bispecific antibody comprises at least oneantibody selected from the group consisting of: an antibody comprising acomplementarity determining region (CDR) having an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,and combinations thereof; an antibody comprising a complementaritydetermining region (CDR) having an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and combinationsthereof; and an antibody comprising a complementarity determining region(CDR) having an amino acid sequence selected from the group consistingof amino acid sequences SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, and combinations thereof. In someembodiments, both antibodies of the bispecific antibody comprise suchCDRs.

In some embodiments, such a bispecific antibody is selected from thegroup consisting of: a bispecific antibody comprising (i) an antibodycomprising a CDR-H1 having amino acid sequence SEQ ID NO:8, a CDR-H2having amino acid sequence SEQ ID NO:10, a CDR-H3 having amino acidsequence SEQ ID NO:12, a CDR-L1 having amino acid sequence SEQ ID NO:14,a CDR-L2 having amino acid sequence SEQ ID NO:16, and a CDR-L3 havingamino acid sequence SEQ ID NO:18 and (ii) an antibody comprising aCDR-H1 having amino acid sequence SEQ ID NO:28, a CDR-H2 having aminoacid sequence SEQ ID NO:30, a CDR-H3 having amino acid sequence SEQ IDNO:32, a CDR-L1 having amino acid sequence SEQ ID NO:34, a CDR-L2 havingamino acid sequence SEQ ID NO:36, and a CDR-L3 having amino acidsequence SEQ ID NO:38; a bispecific antibody comprising (i) an antibodycomprising a CDR-H1 having amino acid sequence SEQ ID NO:8, a CDR-H2having amino acid sequence SEQ ID NO:10, a CDR-H3 having amino acidsequence SEQ ID NO:12, a CDR-L1 having amino acid sequence SEQ ID NO:14,a CDR-L2 having amino acid sequence SEQ ID NO:16, and a CDR-L3 havingamino acid sequence SEQ ID NO:18 and (ii) an antibody comprising aCDR-H1 having amino acid sequence SEQ ID NO:48, a CDR-H2 having aminoacid sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ IDNO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 havingamino acid sequence SEQ ID NO:56, and a CDR-L3 having amino acidsequence SEQ ID NO:58; and a bispecific antibody comprising (i) anantibody comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, aCDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having aminoacid sequence SEQ ID NO:32, a CDR-L1 having amino acid sequence SEQ IDNO:34, a CDR-L2 having amino acid sequence SEQ ID NO:36, and a CDR-L3having amino acid sequence SEQ ID NO:38 and (ii) an antibody comprisinga CDR-H1 having amino acid sequence SEQ ID NO:48, a CDR-H2 having aminoacid sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ IDNO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 havingamino acid sequence SEQ ID NO:56, and a CDR-L3 having amino acidsequence SEQ ID NO:58.

In some embodiments, such a bispecific antibody comprises (i) anantibody comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, aCDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having aminoacid sequence SEQ ID NO:32, a CDR-L1 having amino acid sequence SEQ IDNO:34, a CDR-L2 having amino acid sequence SEQ ID NO:36, and a CDR-L3having amino acid sequence SEQ ID NO:38 and (ii) an antibody comprisinga CDR-H1 having amino acid sequence SEQ ID NO:48, a CDR-H2 having aminoacid sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ IDNO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 havingamino acid sequence SEQ ID NO:56, and a CDR-L3 having amino acidsequence SEQ ID NO:58.

In some embodiments, such a bispecific antibody comprises an antibodyselected from the group consisting of: an antibody comprising a V_(H)chain comprising amino acid sequence SEQ ID NO:4 and a V_(L) chaincomprising amino acid sequence SEQ ID NO:6; an antibody comprising aV_(H) chain comprising amino acid sequence SEQ ID NO:24 and a V_(L)chain comprising amino acid sequence SEQ ID NO:26; an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:44 anda V_(L) chain comprising amino acid sequence SEQ ID NO:46; andcombinations thereof.

In some embodiments, such a bispecific antibody comprises an amino acidsequence selected from the group consisting of amino acid sequences SEQID NO:2, SEQ ID NO:22, SEQ ID NO:42, and combinations thereof.

In some embodiments, such a bispecific antibody is selected from thegroup consisting of: a bispecific antibody comprising an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:4 anda V_(L) chain comprising amino acid sequence SEQ ID NO:6 and an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:24 anda V_(L) chain comprising amino acid sequence SEQ ID NO:26; a bispecificantibody comprising an antibody comprising a V_(H) chain comprisingamino acid sequence SEQ ID NO:4 and a V_(L) chain comprising amino acidsequence SEQ ID NO:6 and an antibody comprising a V_(H) chain comprisingamino acid sequence SEQ ID NO:44 and a V_(L) chain comprising amino acidsequence SEQ ID NO:46; and a bispecific antibody comprising an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:24 anda V_(L) chain comprising amino acid sequence SEQ ID NO:26; and anantibody comprising a V_(H) chain comprising amino acid sequence SEQ IDNO:44 and a V_(L) chain comprising amino acid sequence SEQ ID NO:46.

In some embodiments, such a bispecific antibody is selected from thegroup consisting of: a bispecific antibody comprising an antibodycomprising amino acid sequence SEQ ID NO:2 and an antibody comprisingamino acid sequence SEQ ID NO:22; a bispecific antibody comprising anantibody comprising amino acid sequence SEQ ID NO:2 and an antibodycomprising amino acid sequence SEQ ID NO:42; and a bispecific antibodycomprising an antibody comprising amino acid sequence SEQ ID NO:22 andan antibody comprising amino acid sequence SEQ ID NO:42.

In some embodiments, such a bispecific antibody comprises an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:24 anda V_(L) chain comprising amino acid sequence SEQ ID NO:26 and anantibody comprising a V_(H) chain comprising amino acid sequence SEQ IDNO:44 and a V_(L) chain comprising amino acid sequence SEQ ID NO:46.

In some embodiments, such a bispecific antibody comprises an antibodycomprising amino acid sequence SEQ ID NO:22 and an antibody comprisingamino acid sequence SEQ ID NO:42.

In some embodiments, such a bispecific antibody comprises an Fc domain.

In some embodiments, such an Fc domain comprises an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:102, AND SEQ ID NO:104.

In some embodiments, such a bispecific antibody comprises amino acidsequences SEQ ID NO:22, SEQ ID NO:94, and SEQ ID NO:42.

In some embodiment, such a bispecific antibody comprises amino acidsequence SEQ ID NO:96.

The disclosure provides a human antibody comprising a variable domain,wherein the variable domain is selected from the group consisting of avariable domain comprising the identifying characteristics of humananti-dengue virus antibody m366, a variable domain comprising theidentifying characteristics of human anti-dengue virus antibody m366.6,a variable domain comprising the identifying characteristics of humananti-dengue virus antibody m360, and a variable domain comprising theidentifying characteristics of human anti-dengue virus antibody m360.6.

The disclosure provides a human antibody comprising the identifyingcharacteristics of a human anti-dengue virus antibody selected from thegroup consisting of human anti-dengue virus antibody m366, humananti-dengue virus antibody m366.6, human anti-dengue virus antibodym360, and human anti-dengue virus antibody m360.6.

The disclosure provides a human antibody comprising a variable domainand a constant domain, wherein the human antibody is selected from thegroup consisting of a human antibody having the identifyingcharacteristics of human anti-dengue virus antibody m366, a humanantibody having the identifying characteristics of human anti-denguevirus antibody m366.6, a human antibody having the identifyingcharacteristics of human anti-dengue virus antibody m360, and a humanantibody having the identifying characteristics of human anti-denguevirus antibody m360.6.

The disclosure provides a human anti-dengue virus antibody selected fromthe group consisting of human anti-dengue virus antibody m366, humananti-dengue virus antibody m366.6, human anti-dengue virus antibodym360, and human anti-dengue virus antibody m360.6.

The disclosure provides a bispecific human antibody comprising anantibody comprising a variable domain, wherein the variable domain isselected from the group consisting of a variable domain comprising theidentifying characteristics of human anti-dengue virus antibody m366, avariable domain comprising the identifying characteristics of humananti-dengue virus antibody m366.6, a variable domain comprising theidentifying characteristics of human anti-dengue virus antibody m360.6,and combinations thereof.

The disclosure provides a bispecific antibody comprising an antibodycomprising the identifying characteristics of a human anti-dengue virusantibody selected from the group consisting of human anti-dengue virusantibody m366, human anti-dengue virus antibody m366.6, humananti-dengue virus antibody m360.6, and combinations thereof.

The disclosure provides a bispecific antibody comprising an antibodycomprising a variable domain and a constant domain, wherein the antibodyis selected from the group consisting of a human antibody having theidentifying characteristics of human anti-dengue virus antibody m366, ahuman antibody having the identifying characteristics of humananti-dengue virus antibody m366.6, a human antibody having theidentifying characteristics of human anti-dengue virus antibody m360.6,and combinations thereof.

The disclosure provides a bispecific antibody comprising an antibodyselected from the group consisting of human anti-dengue virus antibodym366, human anti-dengue virus antibody m366.6, human anti-dengue virusantibody m360.6, and combinations thereof.

The disclosure provides human anti-dengue virus antibody m366.

The disclosure provides human anti-dengue virus antibody m366.6.

The disclosure provides human anti-dengue virus antibody m360.6.

The disclosure provides human anti-dengue virus antibody m360.

The disclosure provides a bispecific antibody comprising humananti-dengue virus antibody m366 and human anti-dengue virus antibodym366.6.

The disclosure provides a bispecific antibody comprising humananti-dengue virus antibody m366 and human anti-dengue virus antibodym360.6.

The disclosure provides a bispecific antibody comprising humananti-dengue virus antibody m366.6 and human anti-dengue virus antibodym360.6.

The disclosure provides a bispecific antibody having the identifyingcharacteristics of bispecific anti-dengue virus antibody m3666.

The disclosure provides bispecific anti-dengue virus antibody m3666.

The disclosure provides a human antibody that binds an epitope selectedfrom the group consisting of the epitope which human anti-dengue virusantibody m366 binds, the epitope which human anti-dengue virus antibodym366.6 binds, and the epitope which human anti-dengue virus antibodym360.6 binds.

The disclosure provides a human anti-dengue virus antibody, wherein avariable domain of the antibody has a three-dimensional structuresimilar to that of an antibody selected from the group consisting ofhuman anti-dengue virus antibody m366, human anti-dengue virus antibodym366.6, and human anti-dengue virus antibody m360.6.

The disclosure provides a human anti-dengue virus antibody, wherein CDRsof the antibody are positioned in a three-dimensional structure similarto the positioning of the CDRs of an antibody selected from the groupsconsisting of human anti-dengue virus antibody m366, human anti-denguevirus antibody m366.6, and human anti-dengue virus antibody m360.6.

The disclosure provides a human anti-dengue virus antibody, wherein avariable domain of the antibody contacts at least amino acid residues15, 16, 17, 18, 19, 20, 21, 35, 37, 71, 93, and 95 of amino acidsequence SEQ ID NO:82.

The disclosure provides a human anti-dengue virus antibody of theembodiments produced by a method selected from the group consisting ofrecombinant production and chemical synthesis.

The disclosure provides a nucleic acid molecule that encodes a humananti-dengue virus antibody of the embodiments. Also provided is arecombinant vector comprising such a nucleic acid molecule. Alsoprovided is a recombinant molecule comprising such a nucleic acidmolecule. Also provided is a recombinant cell comprising such arecombinant molecule.

The disclosure provides a pharmaceutical composition comprising a humananti-dengue virus antibody of the embodiments and a pharmaceuticallyacceptable carrier.

In some embodiments, such a pharmaceutical composition comprises onehuman anti-dengue virus antibody of the embodiments and apharmaceutically acceptable carrier.

In some embodiments, such a pharmaceutical composition comprises atleast two human anti-dengue virus antibodies of the embodiments and apharmaceutically acceptable carrier.

In some embodiments, such a pharmaceutical composition comprises atleast one antibody selected from the group consisting of humananti-dengue virus antibody m366, human anti-dengue virus antibodym366.6, human anti-dengue virus antibody m360.6, human anti-dengue virusantibody m360, human anti-dengue virus antibody m3666, and combinationsthereof.

The disclosure provides a treatment for dengue virus infectioncomprising a human anti-dengue virus antibody of the embodiments and apharmaceutically acceptable carrier.

The disclosure provides a preventative composition against dengue virusinfection comprising a human anti-dengue virus antibody of theembodiments and a pharmaceutically acceptable carrier.

The disclosure provides a method to protect a subject from dengue virusinfection, the method comprising administering to the subject a humananti-dengue virus antibody of the embodiments and a pharmaceuticallyacceptable carrier. In some embodiments, the subject is a primate. Insome embodiments, the subject is a human.

The disclosure provides use of a human anti-dengue virus antibody of theembodiments, or a pharmaceutical composition thereof, to protect asubject from dengue virus infection.

The disclosure provides use of a human anti-dengue virus antibody of theembodiments in the manufacture of a medicament for the protection of asubject from dengue virus infection.

The disclosure provides a method to produce a human anti-dengue virusantibody of the embodiments, the method comprising: (a) screening ayeast display human antibody library for a human antibody cross-reactivewith dengue virus (DENV) serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4 in the presence of a non-neutralizing DENV envelopedomain III protein; and (b) isolating a clone expressing such anantibody. In some embodiments, the non-neutralizing DENV envelope domainIII protein has a mutation at a residue corresponding to amino acidresidue 20 of amino acid sequence SEQ ID NO:82. In some embodiments, themutation is a K310E point mutation, wherein the mutation is at aminoacid residue 20 of amino acid sequence SEQ ID NO:82.

The disclosure provides a method to produce a human anti-dengue virusantibody of the embodiments, the method comprising: (a) culturing arecombinant cell encoding the antibody; and (b) recovering the antibody.

The disclosure provides a diagnostic kit comprising a human anti-denguevirus antibody of the embodiments.

The disclosure provides a method to diagnose dengue virus infection in asubject comprising: (a) exposing a human anti-dengue virus antibody ofthe embodiments to the subject or to a sample collected from thesubject; and (b) detecting complex formation between the antibody and anepitope in the subject or in the sample, wherein complex formationindicates that the subject is infected with dengue virus.

The disclosure provides a protein comprising an epitope that binds to anantibody selected from the group consisting of an antibody having theidentifying characteristics of human anti-dengue virus antibody m366, anantibody having the identifying characteristics of human anti-denguevirus antibody m366.6, an antibody having the identifyingcharacteristics of human anti-dengue virus antibody m360.6, and anantibody having the identifying characteristics of human anti-denguevirus antibody m3666. In some embodiments, the antibody is selected fromthe group consisting of human anti-dengue virus antibody m366, humananti-dengue virus antibody m366.6, human anti-dengue virus antibodym360.6, and bispecific anti-dengue virus antibody m3666.

The disclosure provides a protein comprising an epitope that elicitsproduction of an antibody selected from the group consisting of anantibody with identifying characteristics of human anti-dengue virusantibody m366, an antibody having the identifying characteristics ofhuman anti-dengue virus antibody m366.6, and an antibody having theidentifying characteristics of human anti-dengue virus antibody m360.6.In some embodiments, the antibody is selected from the group consistingof human anti-dengue virus antibody m366, human anti-dengue virusantibody m366.6, human anti-dengue virus antibody m360.6, and bispecificanti-dengue virus antibody m3666.

In some embodiments, the antibody is selected from the group consistingof: an antibody comprising a CDR-H1 having amino acid sequence SEQ IDNO:8, a CDR-H2 having amino acid sequence SEQ ID NO:10, a CDR-H3 havingamino acid sequence SEQ ID NO:12, a CDR-L1 having amino acid sequenceSEQ ID NO:14, a CDR-L2 having amino acid sequence SEQ ID NO:16, and aCDR-L3 having amino acid sequence SEQ ID NO:18; an antibody comprising aCDR-H1 having amino acid sequence SEQ ID NO:28, a CDR-H2 having aminoacid sequence SEQ ID NO:30, a CDR-H3 having amino acid sequence SEQ IDNO:32, a CDR-L1 having amino acid sequence SEQ ID NO:34, a CDR-L2 havingamino acid sequence SEQ ID NO:36, and a CDR-L3 having amino acidsequence SEQ ID NO:38; and an antibody comprising a CDR-H1 having aminoacid sequence SEQ ID NO:48, a CDR-H2 having amino acid sequence SEQ IDNO:50, a CDR-H3 having amino acid sequence SEQ ID NO:52, a CDR-L1 havingamino acid sequence SEQ ID NO:54, a CDR-L2 having amino acid sequenceSEQ ID NO:56, and a CDR-L3 having amino acid sequence SEQ ID NO:58.

In some embodiments, the antibody is selected from the group consistingof: an antibody comprising an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NO:4, SEQ ID NO:6, andSEQ ID NO:2; an antibody comprising an amino acid sequence selected fromthe group consisting of amino acid sequences SEQ ID NO:24, SEQ ID NO:26,and SEQ ID NO:22; and an antibody comprising an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:44,SEQ ID NO:46, and SEQ ID NO:42.

In some embodiments, the antibody comprises an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:2,SEQ ID NO:22, SEQ ID NO:42, and SEQ ID NO:96.

In some embodiments, the protein comprises amino acid residuescorresponding to amino acid residues 15, 16, 17, 18, 19, 20, 21, 35, 37,71, 93, and 95 of amino acid sequence SEQ ID NO:82, and wherein theprotein comprises a three-dimension structure in which the amino acidresidues are localized in positions similar to the positions ofcorresponding amino acid residues in a natural DENV envelope domain111.2 protein.

The disclosure provides a pharmaceutical composition comprising such anepitope-containing protein of the embodiments and a pharmaceuticallyacceptable carrier.

The disclosure provides a nucleic acid molecule that encodes such anepitope-containing protein of the embodiments. Also provided is arecombinant vector comprising such a nucleic acid molecule. Alsoprovided is a recombinant molecule comprising such a nucleic acidmolecule. Also provided is a recombinant cell comprising such arecombinant molecule.

The disclosure provides a method to produce a protein that binds to anantibody selected from the group consisting of an antibody having theidentifying characteristics of human anti-dengue virus antibody m366, anantibody having the identifying characteristics of human anti-denguevirus antibody m366.6, an antibody having the identifyingcharacteristics of human anti-dengue virus antibody m360.6, and anantibody having the identifying characteristics of human anti-denguevirus antibody m3666, the method comprising: (a) culturing a recombinantcell encoding such a protein; and (b) recovering the protein. In someembodiments, the antibody is selected from the group consisting of humananti-dengue virus antibody m366, human anti-dengue virus antibodym366.6, human anti-dengue virus antibody m360.6, and bispecificanti-dengue virus antibody m3666.

The disclosure provides a method to protect a subject from dengue virusinfection, the method comprising administering to the subject such anepitope-containing protein of the embodiments, or a pharmaceuticalcomposition thereof. In some embodiments, the subject is a primate. Insome embodiments, the subject is a human.

The disclosure provides use of such an epitope-containing protein of theembodiments, or a pharmaceutical composition thereof, to protect asubject from dengue virus infection.

The disclosure provides use of such an epitope-containing protein of theembodiments in the manufacture of a medicament for the protection of asubject from dengue virus infection.

The disclosure provides a diagnostic kit comprising anepitope-containing protein of the embodiments.

The disclosure provides a method to diagnose dengue virus infection in asubject comprising: (a) exposing an epitope-containing protein of theembodiments to the subject or to a sample collected from the subject;and (b) detecting complex formation between the epitope and an antibodyin the subject or in the sample, wherein complex formation indicatesthat the subject is infected with dengue virus.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B compare sequences of the heavy and light chains of m366antibody (having SEQ ID NO:4 and SEQ ID NO:6, respectively) to sequencesof corresponding germline precursors. Numbering is according to the IMGT(International ImMunoGene Tics) information system numbering scheme. Thesix complementarity-determining regions (CDRs) are depicted in boldface. Only mutated residues are shown for the germline sequences.

FIG. 2 provides consensus amino acid sequences for envelope domain IIIproteins from DENV serotype 1 (DENV1) (SEQ ID NO:97), DENV serotype 2(DENV2) (SEQ ID NO:98), DENV serotype 3 (DENV3) (SEQ ID NO:99), and DENVserotype 4 (DENV 4) (SEQ ID NO:100).

FIG. 3 provides results from an ELISA binding assay that demonstratescross-reactive binding of antibody D6, the unique clone isolated fromthe yeast display naïve antibody library, to dengue virus envelopedomain III proteins (also referred to herein as ED3 proteins, ED3s, orDDs) from all 4 DENV serotypes, i.e., DENV serotype 1 (DD1), DENVserotype 2 (DD2), DENV serotype 3 (DD3), and DENV serotype 4 (DD4).

FIGS. 4A and 4B provide results from a neutralization assay thatdemonstrates potent neutralization of dengue viruses from serotype 2 (A)and serotype 3 (B) by m366. Serially diluted antibody m366 was tested induplicates with the final concentration at (from right to left) 200m/ml, 100 m/ml, 50 μg/ml, 25 μg/ml and 12.5 m/ml. Virus samples withoutany antibody were plated in duplicates as control.

FIG. 5 provides a sequence variation comparison of the domain 111.2(i.e., DIII or ED3 of DENV serotype 2) escape mutants and naturallyisolated dengue virus domain IIIs derived from GenBank. The DENVEnv-DIII.2 protein shown in this figure is represented by amino acidsequence SEQ ID NO:82, which is a 105-amino acid protein that spans fromamino acid 291 through amino acid 395 of the 495-amino acid DENVenvelope serotype 2 protein, the amino acid sequence of whichrepresented by SEQ ID NO:81.

FIG. 6 provides a docking model of the complex between DENV Env-DIII.2and scFv m366. The model is a ribbon representation of a complex betweendengue Env-DIII.2 and scFv antibody m366 in which the experimentallyidentified epitope and contacting residues are shown, withcomplementarity determining regions (CDRs) labeled. Three distinct butstructurally proximal epitope regions are labeled with residue numbers(based on the sequence of the entire 495-amino acid DENV envelopeserotype 2 protein (SEQ ID NO:81) at putative locations in the model.

FIG. 7 illustrates that the m366 epitope is at close proximity to orpartially overlapping the dimerization interface between domains II andIII.

FIG. 8 provides a comparison of the variable heavy chains of mouseanti-dengue virus mAb 9F12 (9F12VH; SEQ ID NO:83) and human anti-denguevirus m366 antibody (m366VH; SEQ ID NO:4). The figure also provides acomparison of the variable light chains of mouse anti-dengue virus mAb9F12 (9F12VL; SEQ ID NO:84) and human anti-dengue virus m366 antibody(m366VL; SEQ ID NO:6). The respective CDRs are underlined. In thefigure, asterisks (*) represent identical amino acids between the twosequences, colons (:) represent conserved substitutions between the twosequences, and periods (.) represent semi-conserved substitution betweenthe two sequences.

FIG. 9 provides results from an ELISA binding assay that demonstratescross-reactive binding of m360 to dengue virus envelope domain IIIproteins from three serotypes.

FIG. 10 provides a schematic of human anti-dengue virus bispecificantibody m3666, which comprises human anti-dengue virus antibodiesm360.6 and m366.6 linked by an Fc domain comprising amino acid sequenceSEQ ID NO:94. The term “Fc (mutations)” refers to an Fc region in whichthe two leucines positioned at amino acid residues 21 and 22 of SEQ IDNO:92 have been deleted, as indicated by the two stars.

FIG. 11 illustrates that human anti-dengue virus antibody m366 does notsignificantly enhance Dengue virus infection.

DETAILED DESCRIPTION

The present disclosure describes the novel finding of a humananti-dengue virus antibody that is cross-reactive with all fourserotypes of dengue virus (DENV). Such an antibody can also neutralizeall four DENV serotypes. In one embodiment, such an antibody can beidentified by sequential panning and screening of a naive human antibodylibrary against envelope domain III proteins from all four serotypes.Such an antibody has potential as a therapeutic, and its epitope as avaccine immunogen.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a nucleic acidmolecule refers to one or more nucleic acid molecules. As such, theterms “a”, “an”, “one or more” and “at least one” can be usedinterchangeably. Similarly the terms “comprising”, “including” and“having” can be used interchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

Human Anti-Dengue Virus Antibodies

The disclosure provides a human anti-dengue virus antibody thatcross-reacts with envelope proteins from all four DENV serotypes; suchan antibody can also neutralize all four serotypes. One embodiment ofsuch an antibody is antibody m366, which was identified by sequentialantigen panning of antibody libraries derived from healthy individualsagainst the DENV envelope domain III, combined with depletion by entrydefective envelope mutants and subsequent in vitro maturation. Humanantibody m366 binds with high affinity to DENV envelope proteins of andneutralizes all four serotypes. The inventors localized the m366 epitopeby computational analysis of docking models of m366 to domain III andsequence analysis of domain III variants. Antibody m366 has a total of 4and 10 amino acid changes, respectively, from the closest VH and Vκgermline gene products; see FIG. 1. Additional embodiments of such anantibody are antibody m366.6, antibody m360.6, and bispecific antibodym3666. Antibody m360, from which antibody m360.6 was derived,cross-reacts with envelope proteins from DENV serotype 1,

DENV serotype 2, and DENV serotype 3. These antibodies representpromising candidate therapeutics and diagnostics. Appropriately designedvaccine immunogens containing at least one of the epitopes bound bym366, m366.6, m360.6, m360, or m3666, respectively, could elicitantibodies with potent cross-reactive neutralizing activity.

The disclosure provides a human anti-dengue virus antibody that binds toan envelope protein of dengue virus, wherein the antibody iscross-reactive with dengue virus (DENV) serotype 1 envelope protein,DENV serotype 2 envelope protein, DENV serotype 3 envelope protein, andDENV serotype 4 envelope protein. As used herein, a human anti-denguevirus antibody is an antibody that binds to dengue virus. An antibody ofthe embodiments binds to a DENV envelope protein. A human anti-denguevirus antibody on the embodiments is an isolated antibody in that it isnot in the presence of its natural milieu. Although it is a humanantibody, it has been isolated from a library comprising humanantibodies than having been isolated from a human subject. Such anantibody can be subject to affinity maturation. One embodiment is ahuman anti-dengue virus antibody that is not isolated from a mammaliansubject. One embodiment is a human anti-dengue virus antibody that isnot isolated from a primate subject. One embodiment is a humananti-dengue virus antibody that is not isolated from a human subj ect.

An antibody of the embodiments is a monoclonal antibody. As used herein,a monoclonal antibody is an antibody produced by a group of identicalcells, all of which were produced from a single cell by repetitivecellular replication. That is, the clone of cells only produces a singleantibody species. While a monoclonal antibody can be produced usinghybridoma production technology, other production methods can also beused, such as those described herein and those known to those skilled inthe art.

As used herein, an antibody refers to a full-length antibody or afragment thereof, wherein the fragment retains the ability to bind to anenvelope protein of dengue virus (i.e., the fragment retains theantibody binding domain, or paratope, of the full-length antibody) andretains the ability to cross-react with envelope proteins from all fourDENV serotypes. Methods to detect and measure antibody binding to anenvelope protein of dengue virus are disclosed herein and are known tothose skilled in the art. Examples of an antibody of the embodimentsinclude, but are not limited to, a full-length antibody (i.e., acomplete antibody, or immunoglobulin, having two full-length heavychains and two full-length light chains), a full-length soluble antibody(i.e., a complete antibody except that it lacks a transmembrane domain),a monospecific antibody, a bispecific antibody, a multi-specificantibody, a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, asingle domain heavy chain antibody, a single domain light chainantibody, and a complementarity determining region (CDR). Other antibodyforms and non-antibody proteins that bind specifically to the epitopesbound by antibodies of the embodiments, e.g., antibodies m366, m366.6,m360.6, and m3666, are also included herein.

A monospecific antibody is an antibody that has one type ofantigen-binding domain, or paratope. A bispecific antibody is anantibody has two types of antigen-binding domains, i.e., two types ofparatopes. Monospecific and bispecific antibodies can be full-lengthsoluble antibodies or any fragment thereof that retains the ability tobind to a DENV envelope protein. One embodiment of a bispecific antibodyis an antibody that has two paratopes, each of which binds to adifferent DENV epitope that is cross-reactive with all four DENVserotypes. One embodiment is a multi-specific antibody, such as anantibody that is bispecific, trispecific, or has specificity for four,five, or more antigens.

In some embodiments, an antibody of the disclosure does not enhanceDengue virus infection. As used herein, the phrase “does not enhanceDengue virus infection” means that the antibody does not significantlyenhance Dengue virus infection (i.e., antibody-dependent enhancement ofinfection (ADE)) in a cell or when administered to a subject, and thecell or subject is exposed to Dengue virus. That is, a cell or subjectadministered an antibody of the embodiments exhibits less than about50%, less than about 20%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2%, less than about1% enhancement of infection when exposed to Dengue virus compared toenhancement of infection exhibited by other antibodies or as a result ofinfection after induction of a humoral immune response against aprevious Dengue virus infection. This characteristic is surprisingbecause typically an induced humoral immune response to one DENVserotype as a result of infection, or an anti-Dengue virus antibody, canenhance the infection and disease processes brought about by asubsequent infection with another DENV serotype; see, for example,Beltramello et al., ibid. Methods to measure ADE are known to thoseskilled in the art.

An antibody of the embodiments can be of any isotype that protects asubject from dengue virus. One embodiment is an antibody of an isotypeselected from IgA, IgD, IgE, IgG, and IgM. One embodiment is an antibodyof an isotype selected from IgG and IgM. One embodiment is an IgAantibody. One embodiment is an IgD antibody. One embodiment is an IgEantibody. One embodiment is an IgG antibody. One embodiment is an IgMantibody.

The disclosure provides a human anti-dengue virus antibody that iscross-reactive with the four known serotypes of dengue virus in that itis cross-reactive with DENV serotype 1 envelope protein, DENV serotype 2envelope protein, DENV serotype 3 envelope protein, and DENV serotype 4envelope protein. It is to be appreciated that one or additional DENVserotypes are yet to be discovered. As such, one embodiment is a humananti-dengue virus antibody that is cross-reactive with envelope proteinsof serotype 1, serotype 2, serotype 3, and serotype 4 and with anenvelope protein from one or more additional DENV serotypes. It is alsoto be appreciated that DENV serotypes 1, 2, 3 and 4 each include severalgenotypes. As such, the disclosure provides a human anti-dengue virusantibody that cross-reacts with envelope proteins of DENV genotypes ofserotype 1, serotype 2, serotype 3, and serotype 4.

One embodiment of the disclosure is a human anti-dengue virus antibodythat binds to a receptor binding domain of a DENV envelope protein,wherein the antibody is cross-reactive with the receptor binding domainof DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype4.

One embodiment of the disclosure is a human anti-dengue virus antibodythat binds to domain III of a DENV envelope protein (also referred toherein as an envelope domain III protein, an envelope protein domainIII, or a domain 3 of a DENV envelope protein), wherein the antibody iscross-reactive with envelope domain III protein of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4. Without being bound bytheory, it is believed that that domain III contains the DENV receptorbinding domain that is associated with viral entry, the first step ofcellular infection.

One embodiment of the disclosure is a human anti-dengue virus antibodythat neutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3, andDENV serotype 4. As used herein, the ability of an antibody toneutralize a DENV serotype refers to the antibody's ability to reducethe ability of a dengue virus serotype to infect a cell. One embodimentis a human anti-dengue virus antibody that neutralizes all four DENVserotypes at an IC₅₀ value of less than about 100 micrograms (pig) permilliliter (ml), of less than about 50 μg/ml, or of less than about 25μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about25 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about20 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about15 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about12.5 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about10 μg/ml, of less than about 9 μg/ml, of less than about 8 μg/ml, ofless than about 7 μg/ml, of less than about 6 μg/ml, of than about 5μg/ml, of less than about 4 μg/ml, of less than about 3 μg/ml, of lessthan about 2 μg/ml, or of less than about 1 μg/ml. One embodiment is ahuman anti-dengue virus antibody that neutralizes all four DENVserotypes at an IC₅₀ value of less than about 0.9 μg/ml, of less thanabout 0.8 μg/ml, of less than about 0.7 μg/ml, of less than about 0.6μg/ml, of than about 0.5 μg/ml, of less than about 0.4 μg/ml, of lessthan about 0.3 μg/ml, of less than about 0.2 μg/ml, or of less thanabout 0.1 μg/ml. One embodiment is a human anti-dengue virus antibodythat neutralizes all four DENV serotypes at an IC₅₀ value of less thanabout 5 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about1 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about0.5 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at an IC₅₀ value of less than about0.1 μg/ml. One embodiment is a human anti-dengue virus antibody thatneutralizes all four DENV serotypes at IC₅₀ values as reported forscFv-Fc m360.6 herein. One embodiment is a human anti-dengue virusantibody that neutralizes all four DENV serotypes at IC₅₀ values asreported for scFv-Fc m366.6 herein. One embodiment is a humananti-dengue virus antibody that neutralizes all four DENV serotypes atIC₅₀ values as reported for m3666 herein. Methods to detect and measureantibody neutralization titers, such as a plaque reduction assay or areporter gene assay, e.g., a DENV RVP assay, are described herein andare known to those skilled in the art. In one embodiment, a DENV RVPassay as described in Example 9 is used to compare the values obtainedfrom a human anti-dengue virus antibody being tested to values citedabove.

One embodiment of the disclosure is a human anti-dengue virus antibodythat binds to a receptor binding domain of a DENV envelope protein,wherein the antibody is cross-reactive with the receptor binding domainof DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype4, and wherein the antibody neutralizes DENV serotype 1, DENV serotype2, DENV serotype 3, and DENV serotype 4.

One embodiment of the disclosure is a human anti-dengue virus antibodythat binds to domain III of a DENV envelope protein), wherein theantibody is cross-reactive with envelope domain III protein of DENVserotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4, andwherein the antibody neutralizes DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4.

One embodiment is a human anti-dengue virus antibody that binds to eachof DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype4 envelope proteins with a dissociation constant (K_(D)) of no more thanabout 50 nanomolar (nM). One embodiment is a human anti-dengue virusantibody that binds to each of DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4 envelope proteins with a K_(D) of nomore than about 40 nM. One embodiment is a human anti-dengue virusantibody that binds to each of DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4 envelope proteins with a K_(D) of nomore than about 20 nM. One embodiment is a human anti-dengue virusantibody that binds to each of DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4 envelope proteins with a K_(D) of nomore than about 100 nM, of no more than about 50 nM, of no more thanabout 20 nM, of no more than about 10 nM, of no more than about 9 nM, ofno more than about 8 nM, of no more than about 7 nM, of no more thanabout 6 nM, of no more than about 5 nM, of no more than about 4 nM of nomore than about 3 nM of no more than about 2 nM, of no more than about 1nM, of no more than about 0.5 nM, of no more than about 0.2 nM, of nomore than about 0.1 nM, of no more than about 0.05 nM, of no more thanabout 0.02 nM, of no more than about 0.01 nM, or of no more than about0.001 nM. One embodiment is a human anti-dengue virus antibody thatbinds to each of DENV serotype 1, DENV serotype 2, DENV serotype 3, andDENV serotype 4 envelope proteins with a K_(D) of no more than about 1nM. One embodiment is a human anti-dengue virus antibody that binds toeach of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4 envelope proteins with a K_(D) of no more than about 0.5 nM.One embodiment is a human anti-dengue virus antibody that binds to eachof DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype4 envelope proteins with a K_(D) of no more than about 10 picomolar(pM). One embodiment is a human anti-dengue virus antibody that binds tothree DENV serotypes with a K_(D) of no more than about 0.5 nM and bindsthe fourth DENV serotype with a K_(D) of no more than about 40 nM.

The disclosure provides a human anti-dengue virus antibody thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:2. That is, the disclosureprovides a human anti-dengue virus antibody that comprises amino acidsequence SEQ ID NO:4, amino acid sequence SEQ ID NO:6, amino acidsequence SEQ ID NO:2 or an amino acid sequence comprising SEQ ID NO:6and SEQ ID NO:4. One embodiment is an antibody that comprises amino acidsequence SEQ ID NO:2. Amino acid sequence SEQ ID NO:2 is the amino acidsequence of human anti-dengue virus antibody m366, described in moredetail herein. Antibody m366 is a single chain variable fragment (scFv)consisting of a variable heavy chain (V_(H), having SEQ ID NO:4) and avariable light chain (V_(L), having SEQ ID NO:6) joined by a linker (L,having SEQ ID NO:20), the order being V_(H)-L-V_(L).

One embodiment is a human anti-dengue virus antibody comprising anantibody scFv in the order V_(H)-L-V_(L), a non-limiting example ofwhich is an antibody having an amino acid sequence in the order SEQID:4-SEQ ID NO:20-SEQ ID NO:6. Another embodiment is a human anti-denguevirus antibody comprising an antibody scFv in the order V_(L)-L-V_(H), anon-limiting example of which is an antibody having an amino acidsequence in the order SEQ ID:6-SEQ ID NO:20-SEQ ID NO:4.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:4. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:4. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:4. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:4. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:4. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:4. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percent,identical to amino acid sequence SEQ ID NO:4. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:4. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:6. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:6. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:6. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:6. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:6. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:6. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percent,identical to amino acid sequence SEQ ID NO:6. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:6. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof. One embodiment isan antibody that comprises amino acid sequences that are at least 50percent identical to amino acid sequences SEQ ID NO:4 and SEQ ID NO:6.One embodiment is an antibody that comprises amino acid sequences thatare at least 60 percent identical to amino acid sequences SEQ ID NO:4and SEQ ID NO:6. One embodiment is an antibody that comprises amino acidsequences that are at least 70 percent identical to amino acid sequencesSEQ ID NO:4 and SEQ ID NO:6. One embodiment is an antibody thatcomprises amino acid sequences that are at least 80 percent identical toamino acid sequences SEQ ID NO:4 and SEQ ID NO:6. One embodiment is anantibody that comprises amino acid sequences that are at least 90percent identical to amino acid sequences SEQ ID NO:4 and SEQ ID NO:6.One embodiment is an antibody that comprises amino acid sequences thatare at least 95 percent identical to amino acid sequences SEQ ID NO:4and SEQ ID NO:6. One embodiment is an antibody comprising an amino acidsequence that is at least 90 percent, at least 91 percent, at least 92percent, at least 93 percent, at least 94 percent, at least 95 percent,at least 96 percent, at least 97 percent, at least 98 percent, or atleast 99 percent, identical to amino acid sequences SEQ ID NO:4 and SEQID NO:6. One embodiment is an antibody that comprises amino acidsequences SEQ ID NO:4 and SEQ ID NO:6. Each of thee antibodies retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an antibody can bea complete Ig or any fragment thereof.

One embodiment is an antibody that comprises amino acid sequences SEQ IDNO:4 and SEQ ID NO:6 wherein SEQ ID NO:4 and SEQ ID NO:6 are joined by apeptide linker. As used herein, two amino acid sequences that are joinedby a peptide linker refers to a protein in which one amino acid sequenceis joined (i.e., fused by a peptide linkage) to the amino terminus ofthe peptide linker and the other amino acid sequence is joined (i.e.,fused by a peptide linkage) to the carboxyl terminus of the peptidelinker. The amino acid composition and length of a peptide linker of theembodiments is typically such to provide flexibility in order to enablethe two amino acid sequences in combination to retain the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such a peptide linker typicallycomprises at least several glycine and/or serine residues. The abilityto design such a peptide linker is known to those skilled in the art.One embodiment is a peptide linker of about 10 to about 25 amino acidsin length. One embodiment is a peptide linker of 10 to 25 amino acids inlength. One embodiment is a linker comprising amino acid sequence SEQ IDNO:20. One embodiment is a linker comprising amino acid sequence SEQ IDNO:40. One embodiment is a linker comprising amino acid sequence SEQ IDNO:60. One embodiment is a linker comprising amino acid sequence SEQ IDNO:80.

One embodiment is an antibody that comprises a first amino acid sequencethat is at least 50 percent identical to amino acid sequence SEQ ID NO:4and a second amino acid sequence that is at least 50 percent identicalto amino acid sequence SEQ ID NO:6, wherein the first amino acidsequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 60 percent identical to amino acid sequenceSEQ ID NO:4 and a second amino acid sequence that is at least 60 percentidentical to amino acid sequence SEQ ID NO:6, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 70 percent identical to amino acid sequenceSEQ ID NO:4 and a second amino acid sequence that is at least 70 percentidentical to amino acid sequence SEQ ID NO:6, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 80 percent identical to amino acid sequenceSEQ ID NO:4 and a second amino acid sequence that is at least 80 percentidentical to amino acid sequence SEQ ID NO:6, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 90 percent identical to amino acid sequenceSEQ ID NO:4 and a second amino acid sequence that is at least 90 percentidentical to amino acid sequence SEQ ID NO:6, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 95 percent identical to amino acid sequenceSEQ ID NO:4 and a second amino acid sequence that is at least 95 percentidentical to amino acid sequence SEQ ID NO:6, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 90 percent, at least 91 percent, at least 92percent, at least 93 percent, at least 94 percent, at least 95 percent,at least 96 percent, at least 97 percent, at least 98 percent, or atleast 99 percent identical to amino acid sequence SEQ ID NO:4 and asecond amino acid sequence that is at least 90 percent, at least 91percent, at least 92 percent, at least 93 percent, at least 94 percent,at least 95 percent, at least 96 percent, at least 97 percent, at least98 percent, or at least 99 percent identical to amino acid sequence SEQID NO:6, wherein the two amino acid sequences are joined by a peptidelinker. One embodiment is an antibody that comprises amino acidsequences SEQ ID NO:4 and SEQ ID NO:6, wherein the two amino acidsequences are joined by a peptide linker. Each of these antibodiesretains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:2. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:2. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

In some embodiments, an antibody of the disclosure comprises at leastone complementarity determining region (CDR) of an antibody selectedfrom the group consisting of human anti-dengue virus antibody m366,human anti-dengue virus antibody m366.6, and human anti-dengue virusantibody m360.6. Methods to identify CDRs are known to those skilled inthe art. Examples of such methods include those of the IMGT

(International ImMunoGene Tics) information system, and methods taughtby: Kabat et al., 1977, J. Biol. Chem. 252, 6609-6616; Kabat et al.,1991, U.S. Dept. of Health and Human Services, “Sequences of proteins ofimmunological interest”; Chothia et al., 1987, J. Mol. Biol. 196,901-917; and MacCallum et al., 1996, J. Mol. Biol. 262:732-745.

The disclosure provides a human anti-dengue virus antibody that binds todomain III of an envelope protein of dengue virus, wherein the antibodyis cross-reactive with domain III of dengue virus (DENV) serotype 1envelope protein, domain III of DENV serotype 2 envelope protein, domainIII of DENV serotype 3 envelope protein, and domain III of DENV serotype4 envelope protein, and wherein the antibody comprises a CDR-H3 havingan amino acid sequence selected from the group consisting of SEQ IDNO:12, SEQ ID NO:32, and SEQ ID NO:52.

The disclosure also provides a human anti-dengue virus antibody thatcomprises a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, and combinations thereof. One embodiment is anantibody comprising a CDR having amino acid sequence SEQ ID NO:8. Oneembodiment is an antibody comprising a CDR having amino acid sequenceSEQ ID NO:10. One embodiment is an antibody comprising a CDR havingamino acid sequence SEQ ID NO:12. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:14. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:16.One embodiment is an antibody comprising a CDR having amino acidsequence SEQ ID NO:18. One embodiment is an antibody, the CDR-H1 ofwhich comprises SEQ ID NO:8. One embodiment is an antibody, the CDR-H2of which comprises SEQ ID NO:10. One embodiment is an antibody, theCDR-H3 of which comprises SEQ ID NO:12. One embodiment is an antibody,the CDR-L1 of which comprises SEQ ID NO:14. One embodiment is anantibody, the CDR-L2 of which comprises SEQ ID NO:16. One embodiment isan antibody, the CDR-L3 of which comprises SEQ ID NO:18. One embodimentis an antibody having any combination of these six CDRs, e.g.,comprising two of these CDRs, three of these CDRs, four of these CDRs,five of these CDRs, or all six of these CDRs. One embodiment is anantibody comprising a V_(H) having a CDR having an amino acid sequenceselected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQID NO:12. In one embodiment, such a V_(H) comprises an amino acidsequence comprising SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. Oneembodiment is an antibody comprising a V_(L) having a CDR having anamino acid sequence selected from the group consisting of SEQ ID NO:14,SEQ ID NO:16, and SEQ ID NO:18. In one embodiment, such a V_(L)comprises an amino acid sequence comprising SEQ ID NO:14, SEQ ID NO:16,and SEQ ID NO:18. One embodiment is an antibody that comprises CDRshaving amino acid sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, and SEQ ID NO:18. One embodiment is an antibodywherein CDR-H1 has amino acid sequence SEQ ID NO:8, CDR-H2 has aminoacid sequence SEQ ID NO:10, CDR-H3 has amino acid sequence SEQ ID NO:12,CDR-L1 has amino acid sequence SEQ ID NO:14, CDR-L2 has amino acidsequence SEQ ID NO:16, and CDR-L3 has amino acid sequence SEQ ID NO:18.One embodiment is an antibody wherein CDR-H3 has amino acid sequence SEQID NO:12 and CDR-L1 has amino acid sequence SEQ ID NO:14. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

The disclosure provides a human anti-dengue virus antibody thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:22. That is, the disclosureprovides a human anti-dengue virus antibody that comprises amino acidsequence SEQ ID NO:24, amino acid sequence SEQ ID NO:26, amino acidsequence SEQ ID NO:22 or an amino acid sequence comprising SEQ ID NO:26and SEQ ID NO:24. One embodiment is an antibody that comprises aminoacid sequence SEQ ID NO:22. Amino acid sequence SEQ ID NO:22 is theamino acid sequence of human anti-dengue virus antibody m366.6,described in more detail herein. Antibody m366.6 is a single chainvariable fragment (scFv) consisting of a variable heavy chain (V_(H),having SEQ ID NO:24) and a variable light chain (V_(L), having SEQ IDNO:26) joined by a linker (L, having SEQ ID NO:40), the order beingV_(H)-L-V_(L).

One embodiment is a human anti-dengue virus antibody comprising anantibody scFv in the order L-L-V_(L), a non-limiting example of which isan antibody having an amino acid sequence in the order SEQ ID NO:24-SEQID NO:40-SEQ ID NO:26. Another embodiment is a human anti-dengue virusantibody comprising an antibody scFv in the order V_(L)-L-V_(H), anon-limiting example of which is an antibody having an amino acidsequence in the order SEQ ID NO:26-SEQ ID NO:40-SEQ ID NO:24.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:24. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:24. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:24. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:24. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:24. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:24. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:24. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:24. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:26. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:26. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:26. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:26. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:26. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:26. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:26. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:26. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody that comprises amino acid sequences thatare at least 50 percent identical to amino acid sequences SEQ ID NO:24and SEQ ID NO:26. One embodiment is an antibody that comprises aminoacid sequences that are at least 60 percent identical to amino acidsequences SEQ ID NO:24 and SEQ ID NO:26. One embodiment is an antibodythat comprises amino acid sequences that are at least 70 percentidentical to amino acid sequences SEQ ID NO:24 and SEQ ID NO:26. Oneembodiment is an antibody that comprises amino acid sequences that areat least 80 percent identical to amino acid sequences SEQ ID NO:24 andSEQ ID NO:26. One embodiment is an antibody that comprises amino acidsequences that are at least 90 percent identical to amino acid sequencesSEQ ID NO:24 and SEQ ID NO:26. One embodiment is an antibody thatcomprises amino acid sequences that are at least 95 percent identical toamino acid sequences SEQ ID NO:24 and SEQ ID NO:26. One embodiment is anantibody comprising an amino acid sequence that is at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical to aminoacid sequences SEQ ID NO:24 and SEQ ID NO:26. One embodiment is anantibody that comprises amino acid sequences SEQ ID NO:24 and SEQ IDNO:26. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

One embodiment is an antibody that comprises amino acid sequences SEQ IDNO:24 and SEQ ID NO:26 wherein SEQ ID NO:224 and SEQ ID NO:26 are joinedby a peptide linker. As used herein, two amino acid sequences that arejoined by a peptide linker refers to a protein in which one amino acidsequence is joined (i.e., fused by a peptide linkage) to the aminoterminus of the peptide linker and the other amino acid sequence isjoined (i.e., fused by a peptide linkage) to the carboxyl terminus ofthe peptide linker. The amino acid composition and length of a peptidelinker of the embodiments is typically such to provide flexibility inorder to enable the two amino acid sequences in combination to retainthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such a peptide linkertypically comprises at least several glycine and/or serine residues. Theability to design such a peptide linker is known to those skilled in theart. One embodiment is a peptide linker of about 10 to about 25 aminoacids in length. One embodiment is a peptide linker of 10 to 25 aminoacids in length. One embodiment is a linker comprising amino acidsequence SEQ ID NO:40. One embodiment is a linker comprising amino acidsequence SEQ ID NO:20. One embodiment is a linker comprising amino acidsequence SEQ ID NO:60. One embodiment is a linker comprising amino acidsequence SEQ ID NO:80.

One embodiment is an antibody that comprises a first amino acid sequencethat is at least 50 percent identical to amino acid sequence SEQ IDNO:24 and a second amino acid sequence that is at least 50 percentidentical to amino acid sequence SEQ ID NO:26, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 60 percent identical to amino acid sequenceSEQ ID NO:24 and a second amino acid sequence that is at least 60percent identical to amino acid sequence SEQ ID NO:26, wherein the firstamino acid sequence and the second amino acid sequence are joined by apeptide linker. One embodiment is an antibody that comprises a firstamino acid sequence that is at least 70 percent identical to amino acidsequence SEQ ID NO:24 and a second amino acid sequence that is at least70 percent identical to amino acid sequence SEQ ID NO:26, wherein thefirst amino acid sequence and the second amino acid sequence are joinedby a peptide linker. One embodiment is an antibody that comprises afirst amino acid sequence that is at least 80 percent identical to aminoacid sequence SEQ ID NO:24 and a second amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:26, whereinthe first amino acid sequence and the second amino acid sequence arejoined by a peptide linker. One embodiment is an antibody that comprisesa first amino acid sequence that is at least 90 percent identical toamino acid sequence SEQ ID NO:24 and a second amino acid sequence thatis at least 90 percent identical to amino acid sequence SEQ ID NO:26,wherein the first amino acid sequence and the second amino acid sequenceare joined by a peptide linker. One embodiment is an antibody thatcomprises a first amino acid sequence that is at least 95 percentidentical to amino acid sequence SEQ ID NO:24 and a second amino acidsequence that is at least 95 percent identical to amino acid sequenceSEQ ID NO:26, wherein the first amino acid sequence and the second aminoacid sequence are joined by a peptide linker. One embodiment is anantibody comprising a first amino acid sequence that is at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical toamino acid sequence SEQ ID NO:24 and a second amino acid sequence thatis at least 90 percent, at least 91 percent, at least 92 percent, atleast 93 percent, at least 94 percent, at least 95 percent, at least 96percent, at least 97 percent, at least 98 percent, or at least 99percent identical to amino acid sequence SEQ ID NO:26, wherein the twoamino acid sequences are joined by a peptide linker. One embodiment isan antibody that comprises amino acid sequences SEQ ID NO:24 and SEQ IDNO:26, wherein the two amino acid sequences are joined by a peptidelinker. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:22. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:22. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:22. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:22. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:22. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:22. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:22. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:22. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is a human anti-dengue virus antibody that comprises aCDR having an amino acid sequence selected from the group consisting ofSEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,SEQ ID NO:38, and combinations thereof. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:28. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:30.One embodiment is an antibody comprising a CDR having amino acidsequence SEQ ID NO:32. One embodiment is an antibody comprising a CDRhaving amino acid sequence SEQ ID NO:34. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:36. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:38.One embodiment is an antibody, the CDR-H1 of which comprises SEQ IDNO:28. One embodiment is an antibody, the CDR-H2 of which comprises SEQID NO:30. One embodiment is an antibody, the CDR-H3 of which comprisesSEQ ID NO:32. One embodiment is an antibody, the CDR-L1 of whichcomprises SEQ ID NO:34. One embodiment is an antibody, the CDR-L2 ofwhich comprises SEQ ID NO:36. One embodiment is an antibody, the CDR-L3of which comprises SEQ ID NO:38. One embodiment is an antibody havingany combination of these six CDRs, e.g., comprising two of these CDRs,three of these CDRs, four of these CDRs, five of these CDRs, or all sixof these CDRs. One embodiment is an antibody comprising a V_(H) having aCDR having an amino acid sequence selected from the group consisting ofSEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32. In one embodiment, such aV_(H) comprises an amino acid sequence comprising SEQ ID NO:28, SEQ IDNO:30, and SEQ ID NO:32. One embodiment is an antibody comprising aV_(L) having a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:38. In oneembodiment, such a V_(L) comprises an amino acid sequence comprising SEQID NO:34, SEQ ID NO:36, and SEQ ID NO:38. One embodiment is an antibodythat comprises CDRs having amino acid sequences SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:38. Oneembodiment is an antibody wherein CDR-H1 has amino acid sequence SEQ IDNO:28, CDR-H2 has amino acid sequence SEQ ID NO:30, CDR-H3 has aminoacid sequence SEQ ID NO:32, CDR-L1 has amino acid sequence SEQ ID NO:34,CDR-L2 has amino acid sequence SEQ ID NO:36, and CDR-L3 has amino acidsequence SEQ ID NO:38. One embodiment is an antibody wherein CDR-H3 hasamino acid sequence SEQ ID NO:32 and CDR-L1 has amino acid sequence SEQID NO:34. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a human anti-dengue virus antibody thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:42. That is, the disclosureprovides a human anti-dengue virus antibody that comprises amino acidsequence SEQ ID NO:44, amino acid sequence SEQ ID NO:46, amino acidsequence SEQ ID NO:42 or an amino acid sequence comprising SEQ ID NO:46and SEQ ID NO:44. One embodiment is an antibody that comprises aminoacid sequence SEQ ID NO:42. Amino acid sequence SEQ ID NO:42 is theamino acid sequence of human anti-dengue virus antibody m360.6,described in more detail herein. Antibody m360.6 is a single chainvariable fragment (scFv) consisting of a variable heavy chain (V_(H),having SEQ ID NO:44) and a variable light chain (V_(L), having SEQ IDNO:46) joined by a linker (L, having SEQ ID NO:60), the order beingV_(H)-L-V_(L).

One embodiment is a human anti-dengue virus antibody comprising anantibody scFv in the order V_(H)-L-V_(L), a non-limiting example ofwhich is an antibody having an amino acid sequence in the order SEQ IDNO:44-SEQ ID NO:60-SEQ ID NO:46. Another embodiment is a humananti-dengue virus antibody comprising an antibody scFv in the orderV_(L)-L-V_(H), a non-limiting example of which is an antibody having anamino acid sequence in the order SEQ ID NO:46-SEQ ID NO:60-SEQ ID NO:44.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:44. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:44. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:44. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:44. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:44. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:44. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:44. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:44. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:46. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:46. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:46. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:46. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:46. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:46. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:46. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:46. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody that comprises amino acid sequences thatare at least 50 percent identical to amino acid sequences SEQ ID NO:44and SEQ ID NO:46. One embodiment is an antibody that comprises aminoacid sequences that are at least 60 percent identical to amino acidsequences SEQ ID NO:44 and SEQ ID NO:46. One embodiment is an antibodythat comprises amino acid sequences that are at least 70 percentidentical to amino acid sequences SEQ ID NO:44 and SEQ ID NO:46. Oneembodiment is an antibody that comprises amino acid sequences that areat least 80 percent identical to amino acid sequences SEQ ID NO:44 andSEQ ID NO:46. One embodiment is an antibody that comprises amino acidsequences that are at least 90 percent identical to amino acid sequencesSEQ ID NO:44 and SEQ ID NO:46. One embodiment is an antibody thatcomprises amino acid sequences that are at least 95 percent identical toamino acid sequences SEQ ID NO:44 and SEQ ID NO:46. One embodiment is anantibody comprising an amino acid sequence that is at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical to aminoacid sequences SEQ ID NO:44 and SEQ ID NO:46. One embodiment is anantibody that comprises amino acid sequences SEQ ID NO:44 and SEQ IDNO:46. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

One embodiment is an antibody that comprises amino acid sequences SEQ IDNO:44 and SEQ ID NO:46 wherein SEQ ID NO:44 and SEQ ID NO:46 are joinedby a peptide linker. As used herein, two amino acid sequences that arejoined by a peptide linker refers to a protein in which one amino acidsequence is joined (i.e., fused by a peptide linkage) to the aminoterminus of the peptide linker and the other amino acid sequence isjoined (i.e., fused by a peptide linkage) to the carboxyl terminus ofthe peptide linker. The amino acid composition and length of a peptidelinker of the embodiments is typically such to provide flexibility inorder to enable the two amino acid sequences in combination to retainthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such a peptide linkertypically comprises at least several glycine and/or serine residues. Theability to design such a peptide linker is known to those skilled in theart. One embodiment is a peptide linker of about 10 to about 25 aminoacids in length. One embodiment is a peptide linker of 10 to 25 aminoacids in length. One embodiment is a linker comprising amino acidsequence SEQ ID NO:60. One embodiment is a linker comprising amino acidsequence SEQ ID NO:20. One embodiment is a linker comprising amino acidsequence SEQ ID NO:40. One embodiment is a linker comprising amino acidsequence SEQ ID NO:80.

One embodiment is an antibody that comprises a first amino acid sequencethat is at least 50 percent identical to amino acid sequence SEQ IDNO:44 and a second amino acid sequence that is at least 50 percentidentical to amino acid sequence SEQ ID NO:46, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 60 percent identical to amino acid sequenceSEQ ID NO:44 and a second amino acid sequence that is at least 60percent identical to amino acid sequence SEQ ID NO:46, wherein the firstamino acid sequence and the second amino acid sequence are joined by apeptide linker. One embodiment is an antibody that comprises a firstamino acid sequence that is at least 70 percent identical to amino acidsequence SEQ ID NO:44 and a second amino acid sequence that is at least70 percent identical to amino acid sequence SEQ ID NO:46, wherein thefirst amino acid sequence and the second amino acid sequence are joinedby a peptide linker. One embodiment is an antibody that comprises afirst amino acid sequence that is at least 80 percent identical to aminoacid sequence SEQ ID NO:44 and a second amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:46, whereinthe first amino acid sequence and the second amino acid sequence arejoined by a peptide linker. One embodiment is an antibody that comprisesa first amino acid sequence that is at least 90 percent identical toamino acid sequence SEQ ID NO:44 and a second amino acid sequence thatis at least 90 percent identical to amino acid sequence SEQ ID NO:46,wherein the first amino acid sequence and the second amino acid sequenceare joined by a peptide linker. One embodiment is an antibody thatcomprises a first amino acid sequence that is at least 95 percentidentical to amino acid sequence SEQ ID NO:44 and a second amino acidsequence that is at least 95 percent identical to amino acid sequenceSEQ ID NO:46, wherein the first amino acid sequence and the second aminoacid sequence are joined by a peptide linker. One embodiment is anantibody comprising a first amino acid sequence that is at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical toamino acid sequence SEQ ID NO:44 and a second amino acid sequence thatis at least 90 percent, at least 91 percent, at least 92 percent, atleast 93 percent, at least 94 percent, at least 95 percent, at least 96percent, at least 97 percent, at least 98 percent, or at least 99percent identical to amino acid sequence SEQ ID NO:46, wherein the twoamino acid sequences are joined by a peptide linker. One embodiment isan antibody that comprises amino acid sequences SEQ ID NO:44 and SEQ IDNO:46, wherein the two amino acid sequences are joined by a peptidelinker. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:42. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:42. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:42. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:42. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:42. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:42. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:42. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:42. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is a human anti-dengue virus antibody that comprises aCDR having an amino acid sequence selected from the group consisting ofSEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:58, and combinations thereof. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:48. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:50.One embodiment is an antibody comprising a CDR having amino acidsequence SEQ ID NO:52. One embodiment is an antibody comprising a CDRhaving amino acid sequence SEQ ID NO:54. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:56. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:58.One embodiment is an antibody, the CDR-H1 of which comprises SEQ IDNO:48. One embodiment is an antibody, the CDR-H2 of which comprises SEQID NO:50. One embodiment is an antibody, the CDR-H3 of which comprisesSEQ ID NO:52. One embodiment is an antibody, the CDR-L1 of whichcomprises SEQ ID NO:54. One embodiment is an antibody, the CDR-L2 ofwhich comprises SEQ ID NO:56. One embodiment is an antibody, the CDR-L3of which comprises SEQ ID NO:58. One embodiment is an antibody havingany combination of these six CDRs, e.g., comprising two of these CDRs,three of these CDRs, four of these CDRs, five of these CDRs, or all sixof these CDRs. One embodiment is an antibody comprising a V_(H) having aCDR having an amino acid sequence selected from the group consisting ofSEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:52. In one embodiment, such aV_(H) comprises an amino acid sequence comprising SEQ ID NO:48, SEQ IDNO:50, and SEQ ID NO:52. One embodiment is an antibody comprising aV_(L) having a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. In oneembodiment, such a V_(L) comprises an amino acid sequence comprising SEQID NO:54, SEQ ID NO:56, and SEQ ID NO:58. One embodiment is an antibodythat comprises CDRs having amino acid sequences SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. Oneembodiment is an antibody wherein CDR-H1 has amino acid sequence SEQ IDNO:48, CDR-H2 has amino acid sequence SEQ ID NO:50, CDR-H3 has aminoacid sequence SEQ ID NO:52, CDR-L1 has amino acid sequence SEQ ID NO:54,CDR-L2 has amino acid sequence SEQ ID NO:56, and CDR-L3 has amino acidsequence SEQ ID NO:58. One embodiment is an antibody wherein CDR-H3 hasamino acid sequence SEQ ID NO:52 and CDR-L1 has amino acid sequence SEQID NO:54. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a human anti-dengue virus antibody thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:62. That is, the disclosureprovides a human anti-dengue virus antibody that comprises amino acidsequence SEQ ID NO:64, amino acid sequence SEQ ID NO:66, amino acidsequence SEQ ID NO:62 or an amino acid sequence comprising SEQ ID NO:66and SEQ ID NO:64. One embodiment is an antibody that comprises aminoacid sequence SEQ ID NO:62. Amino acid sequence SEQ ID NO:62 is theamino acid sequence of human anti-dengue virus antibody m360, describedin more detail herein. Antibody m360 is a single chain variable fragment(scFv) consisting of a variable heavy chain (V_(H), having SEQ ID NO:64)and a variable light chain (V_(L), having SEQ ID NO:66) joined by alinker (L, having SEQ ID NO:80), the order being V_(H)-L-V_(L).

One embodiment is a human anti-dengue virus antibody comprising anantibody scFv in the order V_(H)-L-V_(L), a non-limiting example ofwhich is an antibody having an amino acid sequence in the order SEQ IDNO:64-SEQ ID NO:80-SEQ ID NO:66. Another embodiment is a humananti-dengue virus antibody comprising an antibody scFv in the orderV_(L)-L-V_(H), a non-limiting example of which is an antibody having anamino acid sequence in the order SEQ ID NO:66-SEQ ID NO:80-SEQ ID NO:64.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:64. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:64. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:64. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:64. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:64. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:64. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:64. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:64. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:66. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:66. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:66. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:66. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:66. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:66. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:66. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:66. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is an antibody that comprises amino acid sequences thatare at least 50 percent identical to amino acid sequences SEQ ID NO:64and SEQ ID NO:66. One embodiment is an antibody that comprises aminoacid sequences that are at least 60 percent identical to amino acidsequences SEQ ID NO:64 and SEQ ID NO:66. One embodiment is an antibodythat comprises amino acid sequences that are at least 70 percentidentical to amino acid sequences SEQ ID NO:64 and SEQ ID NO:66. Oneembodiment is an antibody that comprises amino acid sequences that areat least 80 percent identical to amino acid sequences SEQ ID NO:64 andSEQ ID NO:66. One embodiment is an antibody that comprises amino acidsequences that are at least 90 percent identical to amino acid sequencesSEQ ID NO:64 and SEQ ID NO:66. One embodiment is an antibody thatcomprises amino acid sequences that are at least 95 percent identical toamino acid sequences SEQ ID NO:64 and SEQ ID NO:66. One embodiment is anantibody comprising an amino acid sequence that is at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical to aminoacid sequences SEQ ID NO:64 and SEQ ID NO:66. One embodiment is anantibody that comprises amino acid sequences SEQ ID NO:64 and SEQ IDNO:66. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

One embodiment is an antibody that comprises amino acid sequences SEQ IDNO:64 and SEQ ID NO:66 wherein SEQ ID NO:64 and SEQ ID NO:66 are joinedby a peptide linker. As used herein, two amino acid sequences that arejoined by a peptide linker refers to a protein in which one amino acidsequence is joined (i.e., fused by a peptide linkage) to the aminoterminus of the peptide linker and the other amino acid sequence isjoined (i.e., fused by a peptide linkage) to the carboxyl terminus ofthe peptide linker. The amino acid composition and length of a peptidelinker of the embodiments is typically such to provide flexibility inorder to enable the two amino acid sequences in combination to retainthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such a peptide linkertypically comprises at least several glycine and/or serine residues. Theability to design such a peptide linker is known to those skilled in theart. One embodiment is a peptide linker of about 10 to about 25 aminoacids in length. One embodiment is a peptide linker of 10 to 25 aminoacids in length. One embodiment is a linker comprising amino acidsequence SEQ ID NO:80. One embodiment is a linker comprising amino acidsequence SEQ ID NO:20. One embodiment is a linker comprising amino acidsequence SEQ ID NO:40. One embodiment is a linker comprising amino acidsequence SEQ ID NO:60.

One embodiment is an antibody that comprises a first amino acid sequencethat is at least 50 percent identical to amino acid sequence SEQ IDNO:64 and a second amino acid sequence that is at least 50 percentidentical to amino acid sequence SEQ ID NO:66, wherein the first aminoacid sequence and the second amino acid sequence are joined by a peptidelinker. One embodiment is an antibody that comprises a first amino acidsequence that is at least 60 percent identical to amino acid sequenceSEQ ID NO:64 and a second amino acid sequence that is at least 60percent identical to amino acid sequence SEQ ID NO:66, wherein the firstamino acid sequence and the second amino acid sequence are joined by apeptide linker. One embodiment is an antibody that comprises a firstamino acid sequence that is at least 70 percent identical to amino acidsequence SEQ ID NO:64 and a second amino acid sequence that is at least70 percent identical to amino acid sequence SEQ ID NO:66, wherein thefirst amino acid sequence and the second amino acid sequence are joinedby a peptide linker. One embodiment is an antibody that comprises afirst amino acid sequence that is at least 80 percent identical to aminoacid sequence SEQ ID NO:64 and a second amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:66, whereinthe first amino acid sequence and the second amino acid sequence arejoined by a peptide linker. One embodiment is an antibody that comprisesa first amino acid sequence that is at least 90 percent identical toamino acid sequence SEQ ID NO:64 and a second amino acid sequence thatis at least 90 percent identical to amino acid sequence SEQ ID NO:66,wherein the first amino acid sequence and the second amino acid sequenceare joined by a peptide linker. One embodiment is an antibody thatcomprises a first amino acid sequence that is at least 95 percentidentical to amino acid sequence SEQ ID NO:64 and a second amino acidsequence that is at least 95 percent identical to amino acid sequenceSEQ ID NO:66, wherein the first amino acid sequence and the second aminoacid sequence are joined by a peptide linker. One embodiment is anantibody comprising a first amino acid sequence that is at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical toamino acid sequence SEQ ID NO:64 and a second amino acid sequence thatis at least 90 percent, at least 91 percent, at least 92 percent, atleast 93 percent, at least 94 percent, at least 95 percent, at least 96percent, at least 97 percent, at least 98 percent, or at least 99percent identical to amino acid sequence SEQ ID NO:66, wherein the twoamino acid sequences are joined by a peptide linker. One embodiment isan antibody that comprises amino acid sequences SEQ ID NO:64 and SEQ IDNO:66, wherein the two amino acid sequences are joined by a peptidelinker. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

One embodiment is an antibody comprising an amino acid sequence that isat least 50 percent identical to amino acid sequence SEQ ID NO:62. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 60 percent identical to amino acid sequence SEQ ID NO:62. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 70 percent identical to amino acid sequence SEQ ID NO:62. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 80 percent identical to amino acid sequence SEQ ID NO:62. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent identical to amino acid sequence SEQ ID NO:62. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 95 percent identical to amino acid sequence SEQ ID NO:62. Oneembodiment is an antibody comprising an amino acid sequence that is atleast 90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:62. One embodiment is anantibody comprising amino acid sequence SEQ ID NO:62. Each of theseantibodies retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anantibody can be a complete Ig or any fragment thereof.

One embodiment is a human anti-dengue virus antibody that comprises aCDR having an amino acid sequence selected from the group consisting ofSEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76,SEQ ID NO:78, and combinations thereof. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:68. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:70.One embodiment is an antibody comprising a CDR having amino acidsequence SEQ ID NO:72. One embodiment is an antibody comprising a CDRhaving amino acid sequence SEQ ID NO:74. One embodiment is an antibodycomprising a CDR having amino acid sequence SEQ ID NO:76. One embodimentis an antibody comprising a CDR having amino acid sequence SEQ ID NO:78.One embodiment is an antibody, the CDR-H1 of which comprises SEQ IDNO:68. One embodiment is an antibody, the CDR-H2 of which comprises SEQID NO:70. One embodiment is an antibody, the CDR-H3 of which comprisesSEQ ID NO:72. One embodiment is an antibody, the CDR-L1 of whichcomprises SEQ ID NO:74. One embodiment is an antibody, the CDR-L2 ofwhich comprises SEQ ID NO:76. One embodiment is an antibody, the CDR-L3of which comprises SEQ ID NO:78. One embodiment is an antibody havingany combination of these six CDRs, e.g., comprising two of these CDRs,three of these CDRs, four of these CDRs, five of these CDRs, or all sixof these CDRs. One embodiment is an antibody comprising a V_(H) having aCDR having an amino acid sequence selected from the group consisting ofSEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72. In one embodiment, such aV_(H) comprises an amino acid sequence comprising SEQ ID NO:68, SEQ IDNO:70, and SEQ ID NO:72. One embodiment is an antibody comprising aV_(L) having a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:74, SEQ ID NO:76, and SEQ ID NO:78. In oneembodiment, such a V_(L) comprises an amino acid sequence comprising SEQID NO:74, SEQ ID NO:76, and SEQ ID NO:78. One embodiment is an antibodythat comprises CDRs having amino acid sequences SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, and SEQ ID NO:78. Oneembodiment is an antibody wherein CDR-H1 has amino acid sequence SEQ IDNO:68, CDR-H2 has amino acid sequence SEQ ID NO:70, CDR-H3 has aminoacid sequence SEQ ID NO:72, CDR-L1 has amino acid sequence SEQ ID NO:74,CDR-L2 has amino acid sequence SEQ ID NO:76, and CDR-L3 has amino acidsequence SEQ ID NO:78. One embodiment is an antibody wherein CDR-H3 hasamino acid sequence SEQ ID NO:72 and CDR-L1 has amino acid sequence SEQID NO:74. Each of these antibodies retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a human anti-dengue virus antibody of theembodiments that comprises a constant (Fc) domain. In such an embodimentthe antibody domain (which includes the antigen-binding domain) isjoined to an Fc domain. As used herein, an antibody domain joined to anFc domain is an antibody domain that is fused directly to the Fc domainor that is joined to the Fc domain by a linker. In one embodiment, sucha linker is a cleavable linker. In one embodiment, such a linker is anon-cleavable linker. Typically, the Fc domain is joined to the carboxylterminus of the antibody-binding domain by genetic engineeringtechniques. An Fc domain can be a full-length constant domain or anyfragment thereof. Typically, a constant domain is glycosylated and bindsto an Fc gamma receptor. One embodiment is an Fc domain that hasmodified affinity to one or more Fc receptors. One embodiment of thedisclosure is a glycosylated Fc domain that binds to an Fc gammareceptor. One embodiment is an Fc domain that has modified affinity toone or more Fc receptors. One embodiment is an Fc domain that exhibitsreduced binding to an Fc gamma receptor. Such reduction in binding canrange from less than one percent reduction to hundred percent reduction(i.e., from a slight reduction in receptor binding to inability to bindto the receptor). One embodiment is a non-glycosylated Fc domain. Oneembodiment is a non-glycosylated Fc domain that exhibits reduced bindingto an Fc gamma receptor.

The disclosure also provides a human anti-dengue virus antibody of theembodiments that comprises a fusion segment. In such an embodiment, theantibody domain is joined to the fusion segment to form a fusionprotein. As used herein, an antibody domain joined to a fusion segmentis an antibody domain that is fused directly to the fusion segment orthat is joined to the fusion segment by a linker. In one embodiment,such a linker is a cleavable linker. In one embodiment, such a linker isa non-cleavable linker. In one embodiment, the fusion segment is joinedto the carboxyl terminus of the antibody domain. In one embodiment, thefusion segment is joined to the amino terminus of the antibody domain.Typically, the antibody domain and fusion segment are joined by geneticengineering techniques. A fusion segment can provide one or morebenefits to the antibody. Non-limiting examples of fusion segmentsinclude fusion segments that aid in purification (e.g., a His tag, anAvi Tag, a c-Myc tag, a Flag tag, and a HA tag), fusion segments thatare detectable markers (e.g., can be used to detect the antibody in anin vivo, ex vivo, or in vitro diagnostic assay), fusion segments thatare agents to combat dengue virus infection, and fusion segments thatincrease the half-life of the fusion protein.

One embodiment of the disclosure is a human anti-dengue virus antibodythat comprises an Fc domain joined to a fusion segment. As used herein,an Fc domain joined to a fusion segment is an Fc domain that is fuseddirectly to the fusion segment or that is joined to the fusion segmentby a linker. In one embodiment, such a linker is a cleavable linker. Inone embodiment, such a linker is a non-cleavable linker. Typically, thefusion segment is joined to the carboxyl terminus of the Fc domain bygenetic engineering techniques. The disclosure also provides an antibodyin which the fusion segment is joined to the amino terminus of the Fcdomain. Non-limiting examples of fusion segments and their benefits areprovided herein and are known to those skilled in the art.

The disclosure provides a bispecific human anti-dengue virus antibody(also referred to herein as a bispecific anti-dengue virus antibody)that comprises at least one monospecific antibody of the embodiments.Such a bispecific antibody binds to domain III of the envelope proteinof dengue virus and is cross-reactive with domain III of dengue virus(DENV) serotype 1 envelope protein, domain III of DENV serotype 2envelope protein, domain III of DENV serotype 3 envelope protein, anddomain III of DENV serotype 4 envelope protein. Such a bispecificantibody neutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4.

One embodiment is a bispecific anti-dengue virus antibody that comprisestwo monospecific human anti-dengue virus antibodies of the embodiments.In one embodiment, the monospecific antibodies are the same humananti-dengue virus antibody.

In one embodiment, the monospecific antibodies are different humananti-dengue virus antibodies that recognize different DENV virusepitopes. In one embodiment, the bispecific antibody comprises amonospecific human anti-dengue virus antibody of the embodiments and anantibody against another target. In one embodiment, both antibodies ofthe bispecific antibody are cross-reactive with domain III of denguevirus (DENV) serotype 1 envelope protein, domain III of DENV serotype 2envelope protein, domain III of DENV serotype 3 envelope protein, anddomain III of DENV serotype 4 envelope protein.

The disclosure provides a bispecific anti-dengue virus antibody thatneutralizes each of DENV serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4 at an IC_(5o) of less than 25 micrograms per ml(μg/ml), less than 20 μg/ml, less than 15 μg/ml, less than 10 μg/ml,less than 5 μg/ml, less than 1 μg/ml, less than 0.5 μg/ml, less than 0.1μg/ml, less than 0.05 μg/ml, or less than 0.01 μg/ml in a DENV RVP assayas set forth in the Examples. In one embodiment, a bispecificanti-dengue virus antibody neutralizes each of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4 at an IC₅₀ of less than1 μg/ml. In one embodiment, a bispecific anti-dengue virus antibodyneutralizes two of the DENV serotypes at an IC₅₀ of less than 1 μg/mland two of the DENV serotypes at an IC₅₀ of less than 0.1 μg/ml.

The disclosure provides a bispecific anti-dengue virus antibody thatcomprises a human anti-dengue virus antibody that binds to each of DENVserotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4envelope proteins with a dissociation constant (K_(D)) of no more than50 nanomolar (nM), no more than 40 nM, no more than 35 nM, no more than30 nM, no more than 25 nM, no more than 20 nM, no more than 15 nM. nomore than 10 nM, no more than 5 nM, no more than 1 nM, no more than 0.5nM, no more than 0.1 nM, no more than 0.05 nM, no more than 0.01 nM, nomore than 5 pM or no more than 1 pM. One embodiment is a bispecificantibody comprising a human anti-dengue virus antibody that binds toeach of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4 envelope proteins with a K_(D) of no more than 40 nM. Oneembodiment is a bispecific antibody comprising a human anti-dengue virusantibody that binds to each of DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4 envelope proteins with a K_(D) of nomore than 20 nM. One embodiment is a bispecific antibody comprising ahuman anti-dengue virus antibody that binds to each of DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope proteinswith a K_(D) of no more than 1 nM. One embodiment is a bispecificantibody comprising a human anti-dengue virus antibody that binds tothree DENV serotypes with a K_(D) of no more than 0.5 nM and binds thefourth DENV serotype with a K_(D) of no more than 40 nM.

In some embodiments, such a bispecific antibody is selected from thegroup consisting of: a bispecific antibody comprising (i) an antibodycomprising a CDR-H1 having amino acid sequence SEQ ID NO:8, a CDR-H2having amino acid sequence SEQ ID NO:10, a CDR-H3 having amino acidsequence SEQ ID NO:12, a CDR-L1 having amino acid sequence SEQ ID NO:14,a CDR-L2 having amino acid sequence SEQ ID NO:16, and a CDR-L3 havingamino acid sequence SEQ ID NO:18 and (ii) an antibody comprising aCDR-H1 having amino acid sequence SEQ ID NO:28, a CDR-H2 having aminoacid sequence SEQ ID NO:30, a CDR-H3 having amino acid sequence SEQ IDNO:32, a CDR-L1 having amino acid sequence SEQ ID NO:34, a CDR-L2 havingamino acid sequence SEQ ID NO:36, and a CDR-L3 having amino acidsequence SEQ ID NO:38; a bispecific antibody comprising (i) an antibodycomprising a CDR-H1 having amino acid sequence SEQ ID NO:8, a CDR-H2having amino acid sequence SEQ ID NO:10, a CDR-H3 having amino acidsequence SEQ ID NO:12, a CDR-L1 having amino acid sequence SEQ ID NO:14,a CDR-L2 having amino acid sequence SEQ ID NO:16, and a CDR-L3 havingamino acid sequence SEQ ID NO:18 and (ii) an antibody comprising aCDR-H1 having amino acid sequence SEQ ID NO:48, a CDR-H2 having aminoacid sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ IDNO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 havingamino acid sequence SEQ ID NO:56, and a CDR-L3 having amino acidsequence SEQ ID NO:58; and a bispecific antibody comprising (i) anantibody comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, aCDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having aminoacid sequence SEQ ID NO:32, a CDR-L1 having amino acid sequence SEQ IDNO:34, a CDR-L2 having amino acid sequence SEQ ID NO:36, and a CDR-L3having amino acid sequence SEQ ID NO:38 and (ii) an antibody comprisinga CDR-H1 having amino acid sequence SEQ ID NO:48, a CDR-H2 having aminoacid sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ IDNO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 havingamino acid sequence SEQ ID NO:56, and a CDR-L3 having amino acidsequence SEQ ID NO:58.

In some embodiments, such a bispecific antibody comprises (i) anantibody comprising a CDR-H1 having amino acid sequence SEQ ID NO:28, aCDR-H2 having amino acid sequence SEQ ID NO:30, a CDR-H3 having aminoacid sequence SEQ ID NO:32, a CDR-L1 having amino acid sequence SEQ IDNO:34, a CDR-L2 having amino acid sequence SEQ ID NO:36, and a CDR-L3having amino acid sequence SEQ ID NO:38 and (ii) an antibody comprisinga CDR-H1 having amino acid sequence SEQ ID NO:48, a CDR-H2 having aminoacid sequence SEQ ID NO:50, a CDR-H3 having amino acid sequence SEQ IDNO:52, a CDR-L1 having amino acid sequence SEQ ID NO:54, a CDR-L2 havingamino acid sequence SEQ ID NO:56, and a CDR-L3 having amino acidsequence SEQ ID NO:58.

One embodiment is a bispecific anti-dengue virus antibody that comprisesa V_(H) chain comprising amino acid sequence SEQ ID NO:4 and a V_(L)chain comprising amino acid sequence SEQ ID NO:6. One embodiment is abispecific anti-dengue virus antibody that comprises a V_(H) chaincomprising amino acid sequence SEQ ID NO:24 and a V_(L) chain comprisingamino acid sequence SEQ ID NO:26. One embodiment is a bispecificanti-dengue virus antibody that comprises a V_(H) chain comprising aminoacid sequence SEQ ID NO:44 and a V_(L) chain comprising amino acidsequence SEQ ID NO:46. One embodiment is a bispecific anti-dengue virusantibody that comprises amino acid sequence SEQ ID NO:2. One embodimentis a bispecific anti-dengue virus antibody that comprises amino acidsequence SEQ ID NO:22. One embodiment is a bispecific anti-dengue virusantibody that comprises amino acid sequence SEQ ID NO:42.

The disclosure provides a bispecific anti-dengue virus antibody thatcomprises: an antibody comprising a V_(H) chain comprising amino acidsequence SEQ ID NO:4 and a V_(L) chain comprising amino acid sequenceSEQ ID NO:6; and an antibody comprising a V_(H) chain comprising aminoacid sequence SEQ ID NO:24 and a V_(L) chain comprising amino acidsequence SEQ ID NO:26. The disclosure also provides a bispecificanti-dengue virus antibody that comprises: an antibody comprising aV_(H) chain comprising amino acid sequence SEQ ID NO:4 and a V_(L) chaincomprising amino acid sequence SEQ ID NO:6; and an antibody comprising aV_(H) chain comprising amino acid sequence SEQ ID NO:44 and a V_(L)chain comprising amino acid sequence SEQ ID NO:46. The disclosure alsoprovides a bispecific anti-dengue virus antibody that comprises: anantibody comprising a V_(H) chain comprising amino acid sequence SEQ IDNO:24 and a V_(L) chain comprising amino acid sequence SEQ ID NO:26; andan antibody comprising a V_(H) chain comprising amino acid sequence SEQID NO:44 and a V_(L) chain comprising amino acid sequence SEQ ID NO:46.One embodiment is a bispecific antibody comprising an antibodycomprising amino acid sequence SEQ ID NO:2 and an antibody comprisingamino acid sequence SEQ ID NO:22. One embodiment is a bispecificantibody comprising an antibody comprising amino acid sequence SEQ IDNO:2 and an antibody comprising amino acid sequence SEQ ID NO:42. Oneembodiment is a bispecific antibody comprising an antibody comprisingamino acid sequence SEQ ID NO:22 and an antibody comprising amino acidsequence SEQ ID NO:42.

The disclosure also provides a bispecific anti-dengue virus antibodycomprising an Fc domain. In one embodiment, the Fc domain isglycosylated. In one embodiment, the Fc domain is non-glycosylated. Inone embodiment, the Fc domain has modified affinity to one or more Fcreceptors. In one embodiment, the Fc domain exhibits reduced binding toan Fc gamma receptor. One embodiment is an Fc domain flanked by linkers.One embodiment is a bispecific anti-dengue antibody having an Fc domaincomprising amino acid sequence SEQ ID NO:102. One embodiment is abispecific anti-dengue antibody having an Fc domain comprising aminoacid sequence SEQ ID NO:104; such an Fc domain exhibits reduced bindingto an Fc gamma receptor. One embodiment is a bispecific anti-dengueantibody comprising amino acid sequence SEQ ID NO:92; such an Fc domaincomprises amino acid sequence SEQ ID NO:102 flanked by linkers. Oneembodiment is a bispecific anti-dengue antibody comprising amino acidsequence SEQ ID NO:94; such an Fc domain comprises amino acid sequenceSEQ ID NO:104 flanked by linkers.

The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:2, SEQ ID NO:102, and SEQ IDNO:22. The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:2, SEQ ID NO:104, and SEQ IDNO:22. The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:2, SEQ ID NO:92, and SEQ IDNO:22. The disclosure also provides a bispecific anti-dengue virusantibody comprising amino acid sequences SEQ ID NO:2, SEQ ID NO:94, andSEQ ID NO:22. In one embodiment, the bispecific antibody has an aminoacid sequence in the order SEQ ID:2-SEQ ID NO:94-SEQ ID NO:22. In oneembodiment, the bispecific antibody has an amino acid sequence in theorder SEQ ID:22-SEQ ID NO:94-SEQ ID NO:2.

The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:2, SEQ ID NO:102, and SEQ IDNO:42. The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ

ID NO:2, SEQ ID NO:104, and SEQ ID NO:42. The disclosure provides abispecific anti-dengue virus antibody comprising amino acid sequencesSEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:42. The disclosure alsoprovides a bispecific anti-dengue virus antibody comprising amino acidsequences SEQ ID NO:2, SEQ ID NO:94, and SEQ ID NO:42. In oneembodiment, the bispecific antibody has an amino acid sequence in theorder SEQ ID:2-SEQ ID NO:94-SEQ ID NO:42. In one embodiment, thebispecific antibody has an amino acid sequence in the order SEQID:42-SEQ ID NO:94-SEQ ID NO:2.

The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:22, SEQ ID NO:102, and SEQ IDNO:42. The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:22, SEQ ID NO:104, and SEQ IDNO:42. The disclosure provides a bispecific anti-dengue virus antibodycomprising amino acid sequences SEQ ID NO:22, SEQ ID NO:92, and SEQ IDNO:42. The disclosure also provides a bispecific anti-dengue virusantibody comprising amino acid sequences SEQ ID NO:22, SEQ ID NO:94, andSEQ ID NO:42. In one embodiment, the bispecific antibody has an aminoacid sequence in the order SEQ ID:22-SEQ ID NO:94-SEQ ID NO:42. In oneembodiment, the bispecific antibody has an amino acid sequence in theorder SEQ ID:42-SEQ ID NO:94-SEQ ID NO:22. One embodiment is abispecific anti-dengue virus antibody comprising amino acid sequence SEQID NO:96. One embodiment of such a bispecific antibody is depicted inFIG. 10.

One embodiment is a bispecific antibody comprising an amino acidsequence that is at least 50 percent identical to amino acid sequenceSEQ ID NO:96. One embodiment is an antibody comprising an amino acidsequence that is at least 60 percent identical to amino acid sequenceSEQ ID NO:96. One embodiment is an antibody comprising an amino acidsequence that is at least 70 percent identical to amino acid sequenceSEQ ID NO:96. One embodiment is an antibody comprising an amino acidsequence that is at least 80 percent identical to amino acid sequenceSEQ ID NO:96. One embodiment is an antibody comprising an amino acidsequence that is at least 90 percent identical to amino acid sequenceSEQ ID NO:96. One embodiment is an antibody comprising an amino acidsequence that is at least 95 percent identical to amino acid sequenceSEQ ID NO:96. One embodiment is an antibody comprising an amino acidsequence that is at least 90 percent, at least 91 percent, at least 92percent, at least 93 percent, at least 94 percent, at least 95 percent,at least 96 percent, at least 97 percent, at least 98 percent, or atleast 99 percent identical to amino acid sequence SEQ ID NO:96. Oneembodiment is an antibody comprising amino acid sequence SEQ ID NO:96.Each of these bispecific antibodies retains the ability to bind to aDENV envelope protein and to cross-react with envelope proteins from allfour DENV serotypes.

The disclosure provides a bispecific antibody comprising humananti-dengue virus antibody m366. The disclosure provides a bispecificantibody comprising human anti-dengue virus antibody m366.6. Thedisclosure provides a bispecific antibody comprising human anti-denguevirus antibody m360.6. The disclosure provides a bispecific antibodycomprising human anti-dengue virus antibody m360. The disclosureprovides a bispecific antibody comprising human anti-dengue virusantibodies m366 and m366.6. The disclosure provides a bispecificantibody comprising human anti-dengue virus antibodies m366 and m360.6.The disclosure provides a bispecific antibody comprising humananti-dengue virus antibodies m366 and m360. The disclosure provides abispecific antibody comprising human anti-dengue virus antibodies m366.6and m360.6. The disclosure provides a bispecific antibody comprisinghuman anti-dengue virus antibodies m366.6 and m360. The disclosureprovides a bispecific antibody comprising human anti-dengue virusantibodies m360.6 and m360.

The disclosure provides a bispecific antibody comprising humananti-dengue virus antibody m366 and an Fc domain, such as an Fc domaincomprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as anFc domain comprising amino acid sequence SEQ ID NO:94. The disclosureprovides a bispecific antibody comprising human anti-dengue virusantibody m366.6 and an Fc domain, such as an Fc domain comprising aminoacid sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domaincomprising amino acid sequence SEQ ID NO:94. The disclosure provides abispecific antibody comprising human anti-dengue virus antibody m360.6and an Fc domain, such as an Fc domain comprising amino acid sequenceSEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain comprising amino acidsequence SEQ ID NO:94. The disclosure provides a bispecific antibodycomprising human anti-dengue virus antibody m360 and an Fc domain, suchas an Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ IDNO:94, such as an Fc domain comprising amino acid sequence SEQ ID NO:94.

The disclosure provides a bispecific antibody comprising humananti-dengue virus antibodies m366 and m366.6 and an Fc domain, such asan Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ IDNO:94, such as an Fc domain comprising amino acid sequence SEQ ID NO:94.The disclosure provides a bispecific antibody comprising humananti-dengue virus antibodies m366 and m360.6 and an Fc domain, such asan Fc domain comprising amino acid sequence SEQ ID NO:92 or SEQ IDNO:94, such as an Fc domain comprising amino acid sequence SEQ ID NO:94.The disclosure provides a bispecific antibody comprising humananti-dengue virus antibodies m366 and m360 and an Fc domain, such as anFc domain comprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94,such as an Fc domain comprising amino acid sequence SEQ ID NO:94. Thedisclosure provides a bispecific antibody comprising human anti-denguevirus antibodies m366.6 and m360.6 and an Fc domain, such as an Fcdomain comprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, suchas an Fc domain comprising amino acid sequence SEQ ID NO:94. Thedisclosure provides a bispecific antibody comprising human anti-denguevirus antibodies m366.6 and m360 and an Fc domain, such as an Fc domaincomprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as anFc domain comprising amino acid sequence SEQ ID NO:94. The disclosureprovides a bispecific antibody comprising human anti-dengue virusantibodies m360.6 and m360 and an Fc domain, such as an Fc domaincomprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as anFc domain comprising amino acid sequence SEQ ID NO:94.

The disclosure also provides a human anti-dengue virus antibody of theembodiments that comprises a secretory segment (i.e., a secretorysequence) joined to the amino terminus of the antibody domain. Asecretory segment enables an expressed antibody to be secreted from thecell that produces it. Suitable secretory segments include an antibodysecretory segment or any heterologous secretory segment capable ofdirecting the secretion of an antibody of the embodiments. Examples ofsecretory segments include, but are not limited to, tissue plasminogenactivator (t-PA), interferon, interleukin, growth hormone,histocompatibility, viral envelope glycoprotein, and antibody secretorysegments. In one embodiment, the secretory segment is an antibodysecretory segment.

The disclosure provides an antibody comprising a variable domain havingthe identifying characteristics of human anti-dengue virus antibodym366. Identifying characteristics of human anti-dengue virus antibodym366 include (a) derivation from a human antibody, (b) binding to a DENVenvelope domain III protein, (c) cross-reactive with DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4, (d) binding toeach of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4 envelope proteins with a dissociation constant (K_(D)) of nomore than 20 nanomolar (nM), as determined by a Biocore assay as setforth in the Examples, (e) neutralization of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4, and (f) interactionwith amino acid residue 20 of DENV envelope domain 111.2 protein havingSEQ ID NO:82 (i.e., interaction with amino acid residue 310 of DENVenvelope serotype 2 protein having SEQ ID NO:81). In one embodiment,such an antibody also has the identifying characteristic of binding toeach of the DENV serotypes with a K_(D) of no more than 2, 5, 10, and 15nM, respectively, as determined by a Biocore assay as set forth in theExamples. In one embodiment, such an antibody also has the identifyingcharacteristic of neutralizing each of the DENV serotypes at no morethan 25 μg/ml, as determined in a plaque reduction assay as set forth inthe Examples. SEQ ID NO:81 represents the amino acid sequence of SwissProt: P143338.1: DENV envelope serotype 2, except that several aminoacids have been changed to correspond to SEQ ID NO:82: i.e., residue 310has been changed from E to K; residue 337 has been changed from L to F;residue 343 has been changed from D to E; residue 344 has been changedfrom N to K; residue 367 has been changed from V to I; and residue 373has been changed from L to F.

Without being bound by theory, it is believed that antibody m366interacts with amino acid residues 15, 16, 17, 18, 19, 20, 21, 35, 37,71, 93 and 95 of DENV envelope domain 111.2 protein having amino acidsequence SEQ ID NO:82; these residues correspond to amino acid residues305, 306, 307, 308, 309, 310, 311, 325, 327, 361, 383, and 385 of DENVenvelope serotype 2 protein having amino acid sequence SEQ ID NO:81.

One embodiment is a human anti-dengue virus antibody comprising avariable domain having the identifying characteristics of antibody m366.One embodiment is a human anti-dengue virus antibody comprising avariable domain and a constant domain, wherein the antibody has theidentifying characteristics of antibody m366. One embodiment of thedisclosure is human anti-dengue virus antibody m366, also referred toherein as human DENV antibody m366, human antibody m366, antibody m366,and m366. One embodiment of the disclosure is a human anti-dengue virusantibody comprising amino acid sequence SEQ ID NO:2. One embodiment isan antibody that comprises an antibody m366 paratope. One embodiment isan antibody that binds the epitope which antibody m366 binds.

The disclosure provides a human anti-dengue virus antibody, the variabledomain of which has a three-dimensional structure that is similar tothat of antibody m366. Such a three-dimensional structure enablesbinding of such an antibody to the epitope which antibody m366 binds.One embodiment is an antibody in which the CDRs of the antibody arepositioned in a three-dimensional structure similar to the positioningof the CDRs of antibody m366, i.e., such that the antibody can bind tothe epitope which antibody m366 binds. One embodiment is an antibodycomprising a variable domain that contacts at least one of the followingamino acid residues of the DENV envelope domain 111.2 protein havingamino acid sequence SEQ ID NO:82: amino acid residue 15, 16, 17, 18, 19,20, 21, 35, 37, 71, 93 or 95 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contacts atleast one of the following amino acid residues of the DENV envelopeserotype 2 protein having amino acid sequence SEQ ID NO:81: i.e., aminoacid residue 305, 306, 307, 308, 309, 310, 311, 325, 327, 361, 383, or385 of amino acid sequence SEQ ID NO:81.

One embodiment is an antibody comprising a variable domain that contactsamino acid residue 15 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 16 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 17 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 18 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 19 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 20 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 21 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 35 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 37 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 71 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 93 of amino acid sequence SEQ ID NO:82. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 95 of amino acid sequence SEQ ID NO:82. Thedisclosure provides for an antibody comprising a variable domain thatcontacts any combination of amino acid residues 15, 16, 17, 18, 19, 20,21, 35, 37, 71, 93 and 95 of amino acid sequence SEQ ID NO:82. Thedisclosure provides for an antibody comprising a variable domain thatcontacts amino acid residues 71, 93 and 95 of amino acid sequence SEQ IDNO:82. Without being bound by theory, it is believed that these residuesare only contacted by antibodies of the disclosure.

One embodiment is an antibody comprising a variable domain that contactsamino acid residue 305 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 306 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 307 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 308 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 309 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 310 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 311 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 325 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 327 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 361 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 383 of amino acid sequence SEQ ID NO:81. Oneembodiment is an antibody comprising a variable domain that contactsamino acid residue 385 of amino acid sequence SEQ ID NO:81. Thedisclosure provides for an antibody comprising a variable domain thatcontacts amino acid residues 361, 383 and 385 of amino acid sequence SEQID NO:81. Without being bound by theory, it is believed that theseresidues are only contacted by antibodies of the disclosure.

The disclosure provides an antibody comprising a variable domain havingthe identifying characteristics of human anti-dengue virus antibodym366.6. Identifying characteristics of human anti-dengue virus antibodym366.6 include (a) derivation from a human antibody, (b) binding to aDENV envelope domain III protein, (c) cross-reactive with DENV serotype1, DENV serotype 2, DENV serotype 3, and DENV serotype 4, (d) binding toeach of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4 envelope proteins with a dissociation constant (K_(D)) of nomore than 1 nanomolar (nM), as determined by a Biocore assay as setforth in the Examples, (e) neutralization of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4 at an IC₅₀ of less than25 μg/ml, as determined by a DENV RVP assay as set forth in theExamples, and (f) interaction with amino acid residue 20 of DENVenvelope domain 111.2 protein having SEQ ID NO:82 (i.e., interactionwith amino acid residue 310 of DENV envelope serotype 2 protein havingSEQ ID NO:81). In one embodiment, such an antibody also has theidentifying characteristic of neutralizing each of the DENV serotypes atan IC₅₀ of no more than 0.5. 1, 3, and 25 μg/ml, respectively, asdetermined by a DENV RVP assay as set forth in the Examples.

One embodiment is a human anti-dengue virus antibody comprising avariable domain having the identifying characteristics of antibodym366.6. One embodiment is a human anti-dengue virus antibody comprisinga variable domain and a constant domain, wherein the antibody has theidentifying characteristics of antibody m366.6. One embodiment of thedisclosure is human anti-dengue virus antibody m366.6, also referred toherein as human DENV antibody m366.6, human antibody m366.6, antibodym366.6, and m366.6. One embodiment of the disclosure is a humananti-dengue virus antibody comprising amino acid sequence SEQ ID NO:22.One embodiment is an antibody that comprises an antibody m366.6paratope. One embodiment is an antibody that binds the epitope whichantibody m366.6 binds.

The disclosure provides a human anti-dengue virus antibody, the variabledomain of which has a three-dimensional structure that is similar tothat of antibody m366.6. Such a three-dimensional structure enablesbinding of such an antibody to the epitope which antibody m366.6 binds.One embodiment is an antibody in which the CDRs of the antibody arepositioned in a three-dimensional structure similar to the positioningof the CDRs of antibody m366.6, i.e., such that the antibody can bind tothe epitope which antibody m366.6 binds.

The disclosure provides an antibody comprising a variable domain havingthe identifying characteristics of human anti-dengue virus antibodym360.6. Identifying characteristics of human anti-dengue virus antibodym360.6 include (a) derivation from a human antibody, (b) binding to aDENV envelope domain III protein, (c) cross-reactive with DENV serotype1, DENV serotype 2, DENV serotype 3, and DENV serotype 4, (d) binding toeach of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4 envelope proteins with a dissociation constant (K_(D)) of nomore than 40 nanomolar (nM), as determined by a Biocore assay as setforth in the Examples, (e) neutralization of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4 at an IC₅₀ of less than25 μg/ml, as determined by a DENV RVP assay as set forth in theExamples, and (f) interaction with amino acid residue 20 of DENVenvelope domain 111.2 protein having SEQ ID NO:82 (i.e., interactionwith amino acid residue 310 of DENV envelope serotype 2 protein havingSEQ ID NO:81). In one embodiment, such an antibody also has theidentifying characteristic of binding to each of the DENV serotypes witha K_(D) of no more than 1, 1, 1, and 40 nM, respectively, as determinedby a Biocore assay as set forth in the Examples. In one embodiment, suchan antibody also has the identifying characteristic of neutralizing eachof the DENV serotypes at an IC₅₀ of no more than 2, 5, 15, and 25 μg/ml,respectively, as determined by a DENV RVP assay as set forth in theExamples.

One embodiment is a human anti-dengue virus antibody comprising avariable domain having the identifying characteristics of antibodym360.6. One embodiment is a human anti-dengue virus antibody comprisinga variable domain and a constant domain, wherein the antibody has theidentifying characteristics of antibody m360.6. One embodiment of thedisclosure is human anti-dengue virus antibody m360.6, also referred toherein as human DENV antibody m360.6, human antibody m360.6, antibodym360.6, and m360.6. One embodiment of the disclosure is a humananti-dengue virus antibody comprising amino acid sequence SEQ ID NO:42.One embodiment is an antibody that comprises an antibody m360.6paratope. One embodiment is an antibody that binds the epitope whichantibody m360.6 binds.

The disclosure provides a human anti-dengue virus antibody, the variabledomain of which has a three-dimensional structure that is similar tothat of antibody m360.6. Such a three-dimensional structure enablesbinding of such an antibody to the epitope which antibody m360.6 binds.One embodiment is an antibody in which the CDRs of the antibody arepositioned in a three-dimensional structure similar to the positioningof the CDRs of antibody m360.6, i.e., such that the antibody can bind tothe epitope which antibody m360.6 binds.

The disclosure provides an antibody comprising a variable domain havingthe identifying characteristics of human anti-dengue virus antibodym360. Identifying characteristics of human anti-dengue virus antibodym360 include (a) derivation from a human antibody, (b) binding to a DENVenvelope domain III protein, (c) cross-reactive with DENV serotype 1,DENV serotype 2, and DENV serotype 3, (d) binding to each of DENVserotype 1, DENV serotype 2, and DENV serotype 3 envelope proteins witha dissociation constant (K_(D)) of no more than 10 nM nanomolar (nM),(e) neutralization of three serotypes of DENV, and (f) interaction withamino acid residue 20 of DENV envelope domain 111.2 protein having SEQID NO:82 (i.e., interaction with amino acid residue 310 of DENV envelopeserotype 2 protein having SEQ ID NO:81). In one embodiment, such anantibody also has the identifying characteristic of binding to each ofthe DENV serotypes with a K_(D) of no more than 0.1, 10, 10, and 100 nM,respectively, as determined by a Biocore assay as set forth in theExamples.

One embodiment is a human anti-dengue virus antibody comprising avariable domain having the identifying characteristics of antibody m360.One embodiment is a human anti-dengue virus antibody comprising avariable domain and a constant domain, wherein the antibody has theidentifying characteristics of antibody m360. One embodiment of thedisclosure is human anti-dengue virus antibody m360, also referred toherein as human DENV antibody m360, human antibody m360, antibody m360,and m360. One embodiment of the disclosure is a human anti-dengue virusantibody comprising amino acid sequence SEQ ID NO:62. One embodiment isan antibody that comprises an antibody m360 paratope. One embodiment isan antibody that binds the epitope which antibody m360 binds.

The disclosure provides a human anti-dengue virus antibody, the variabledomain of which has a three-dimensional structure that is similar tothat of antibody m360. Such a three-dimensional structure enablesbinding of such an antibody to the epitope which antibody m360 binds.One embodiment is an antibody in which the CDRs of the antibody arepositioned in a three-dimensional structure similar to the positioningof the CDRs of antibody m360, i.e., such that the antibody can bind tothe epitope which antibody m360 binds.

The disclosure provides a bispecific antibody having the identifyingcharacteristics of human anti-dengue virus antibody m3666. Identifyingcharacteristics of bispecific anti-dengue virus antibody m3666 include(a) comprising human anti-dengue virus antibodies having the identifyingcharacteristics of antibodies m360.6 and m366.6, (b) binding to a DENVenvelope domain III protein, (c) cross-reactive with DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4, (d) binding toeach of DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4 envelope proteins with a dissociation constant (K_(D)) of nomore than 40 nanomolar (nM), as determined by a Biocore assay as setforth in the Examples, (e) neutralization of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4 at an IC₅₀ of less than1 μg/ml, as determined by a DENV RVP assay as set forth in the Examples,and (f) interaction with amino acid residue 20 of DENV envelope domain111.2 protein having SEQ ID NO:82 (i.e., interaction with amino acidresidue 310 of DENV envelope serotype 2 protein having SEQ ID NO:81). Inone embodiment, such a bispecific antibody also has the identifyingcharacteristic of neutralizing two of the DENV serotypes at an IC₅₀ ofless than 1 μg/ml and two of the DENV serotypes at an IC₅₀ of less than0.1 μg/ml, as determined by a DENV RVP assay as set forth in theExamples.

One embodiment of the disclosure is bispecific anti-dengue virusantibody m3666, also referred to herein as bispecific DENV antibodym3666, bispecific antibody m3666, antibody m3666, and m3666. Oneembodiment of the disclosure is a bispecific anti-dengue virus antibodycomprising amino acid sequence SEQ ID NO:96. One embodiment is anantibody that comprises antibody m3666 paratopes. One embodiment is anantibody that binds the epitopes which antibody m3666 binds.

The disclosure provides a bispecific anti-dengue virus antibody, thevariable domains of which have three-dimensional structures that aresimilar to those of bispecific antibody m3666. Such three-dimensionalstructure enables binding of such a bispecific antibody to the epitopeswhich bispecific antibody m3666 binds. One embodiment is an antibody inwhich the CDRs of the antibody are positioned in a three-dimensionalstructure similar to the positioning of the CDRs of bispecific antibodym3666, i.e., such that the antibody can bind to the epitopes whichantibody m3666 binds.

The disclosure also provides human anti-dengue virus antibody conjugatescomprising an anti-dengue virus antibody of the embodiments conjugatedto an agent.

Any of the human anti-dengue virus antibodies disclosed herein,including monospecific, bispecific and multispecific antibodies can beused to form an antibody-agent conjugate. The agent can be conjugated tothe antibody directly or via a linker using techniques known to thoseskilled in the art. The linker can be cleavable or non-cleavable. Theagent can be a therapeutic agent, a toxic agent, or a detectable marker.Examples of such therapeutic agents (e.g., for use in therapy), toxicagents (such as a cytotoxic agent), or detectable markers (e.g., for useas a diagnostic) are known to those skilled in the art.

Epitope-Containing Proteins

The disclosure provides a protein comprising an epitope that binds to anantibody having the identifying characteristics of human anti-denguevirus antibody m366. Such a protein can have utility as an immunogenagainst dengue virus infection. Identifying characteristics of humananti-dengue virus antibody m366 are disclosed herein. One embodiment isa protein comprising an epitope that binds to an antibody comprisingamino acid sequence SEQ ID NO:2. One embodiment is a protein comprisingan epitope that binds to human anti-dengue virus antibody m366. In oneembodiment, the epitope contacts at least one residue in amino acidsequence SEQ ID NO:2 that corresponds to at least one residue contactedby DENV Env-DIII.2. In one embodiment, the epitope contacts at least oneresidue in amino acid sequence SEQ ID NO:2 that corresponds to at leastone residue contacted by DENV envelope proteins from serotype 1,serotype 2, serotype 3, and serotype 4.

The disclosure provides a protein comprising an epitope that binds to anantibody having the identifying characteristics of human anti-denguevirus antibody m366.6. Such a protein can have utility as an immunogenagainst dengue virus infection. Identifying characteristics of humananti-dengue virus antibody m366.6 are disclosed herein. One embodimentis a protein comprising an epitope that binds to an antibody comprisingamino acid sequence SEQ ID NO:22. One embodiment is a protein comprisingan epitope that binds to human anti-dengue virus antibody m366.6. In oneembodiment, the epitope contacts at least one residue in amino acidsequence SEQ ID NO:22 that corresponds to at least one residue contactedby DENV Env-DIII.2. In one embodiment, the epitope contacts at least oneresidue in amino acid sequence SEQ ID NO:22 that corresponds to at leastone residue contacted by DENV envelope proteins from serotype 1,serotype 2, serotype 3, and serotype 4.

The disclosure provides a protein comprising an epitope that binds to anantibody having the identifying characteristics of human anti-denguevirus antibody m360.6. Such a protein can have utility as an immunogenagainst dengue virus infection. Identifying characteristics of humananti-dengue virus antibody m360.6 are disclosed herein. One embodimentis a protein comprising an epitope that binds to an antibody comprisingamino acid sequence SEQ ID NO:42. One embodiment is a protein comprisingan epitope that binds to human anti-dengue virus antibody m360.6. In oneembodiment, the epitope contacts at least one residue in amino acidsequence SEQ ID NO:42 that corresponds to at least one residue contactedby DENV Env-DIII.2. In one embodiment, the epitope contacts at least oneresidue in amino acid sequence SEQ ID NO:42 that corresponds to at leastone residue contacted by DENV envelope proteins from serotype 1,serotype 2, serotype 3, and serotype 4.

The disclosure provides a protein comprising an epitope that binds to anantibody having the identifying characteristics of human anti-denguevirus antibody m360. Such a protein can have utility as an immunogenagainst dengue virus infection. Identifying characteristics of humananti-dengue virus antibody m360 are disclosed herein. One embodiment isa protein comprising an epitope that binds to an antibody comprisingamino acid sequence SEQ ID NO:62. One embodiment is a protein comprisingan epitope that binds to human anti-dengue virus antibody m360. In oneembodiment, the epitope contacts at least one residue in amino acidsequence in SEQ ID NO:62 that corresponds to at least one residuecontacted by DENV Env-DIII.2. In one embodiment, the epitope contacts atleast one residue in amino acid sequence SEQ ID NO:62 that correspondsto at least one residue contacted by DENV envelope proteins fromserotype 1, serotype 2, and serotype 3.

The disclosure provides a protein comprising an epitope that binds to anantibody having the identifying characteristics of bispecific humananti-dengue virus antibody m3666. Such a protein can have utility as animmunogen against dengue virus infection. Identifying characteristics ofbispecific anti-dengue virus antibody m3666 are disclosed herein. Oneembodiment is a protein comprising an epitope that binds to an antibodycomprising amino acid sequence SEQ ID NO:96. One embodiment is a proteincomprising an epitope that binds to human anti-dengue virus antibodym3666. In one embodiment, the epitope contacts at least one residue inamino acid sequence in SEQ ID NO:96 that corresponds to at least oneresidue contacted by DENV Env-DIII.2. In one embodiment, the epitopecontacts at least one residue in amino acid sequence SEQ ID NO:96 thatcorresponds to at least one residue contacted by DENV envelope proteinsfrom serotype 1, serotype 2, serotype 3, and serotype 4.

One embodiment is a protein comprising an epitope that elicitsproduction of an antibody having the identifying characteristics of ahuman anti-dengue virus antibody selected from the group consisting ofanti-dengue virus antibody m366, anti-dengue virus antibody m366.6,anti-dengue virus antibody m360.6, and anti-dengue virus antibody m360.One embodiment is a protein comprising an epitope that elicitsproduction of an antibody having the identifying characteristics ofhuman anti-dengue virus antibody m366. One embodiment is a proteincomprising an epitope that elicits production of an antibody having theidentifying characteristics of human anti-dengue virus antibody m366.6.One embodiment is a protein comprising an epitope that elicitsproduction of an antibody having the identifying characteristics ofhuman anti-dengue virus antibody m360.6. One embodiment is a proteincomprising an epitope that elicits production of an antibody having theidentifying characteristics of human anti-dengue virus antibody m360.One embodiment is a protein comprising an epitope that elicitsproduction of an antibody that binds to DENV envelope proteins fromserotype 1, serotype 2, serotype 3, and serotype 4. One embodiment is aprotein comprising an epitope that elicits production of an antibodythat neutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3, andDENV serotype 4.

The disclosure provides a protein that comprises at least one amino acidresidue corresponding to at least one of amino acid residues 15, 16, 17,18, 19, 20, 21, 35, 37, 71, 93, and 95 of amino acid sequence SEQ IDNO:82, wherein the protein comprises a three-dimensional structure inwhich the at least one amino acid residue is localized in a positionsimilar to the position of the corresponding at least one amino acidresidue in a natural DENV envelope domain 111.2 protein. The disclosureprovides a protein that comprises amino acid residues corresponding toamino acid residues 15, 16, 17, 18, 19, 20, 21, 35, 37, 71, 93, and 95of amino acid sequence SEQ ID NO:82, wherein the protein comprises athree-dimensional structure in which the amino acid residues arelocalized in positions similar to the positions of the correspondingamino acid residues in a natural DENV envelope domain 111.2 protein.

Nucleic Acid Molecules and Uses Thereof

The disclosure provides a nucleic acid molecule that encodes a humananti-dengue virus antibody of the embodiments. The disclosure alsoprovides a nucleic acid molecule that encodes a protein comprising anepitope that binds to an antibody having the identifying characteristicsof human anti-dengue virus antibody m366. The disclosure also provides anucleic acid molecule that encodes a protein comprising an epitope thatbinds to an antibody having the identifying characteristics of humananti-dengue virus antibody m366.6. The disclosure also provides anucleic acid molecule that encodes a protein comprising an epitope thatbinds to an antibody having the identifying characteristics of humananti-dengue virus antibody m360.6. The disclosure also provides anucleic acid molecule that encodes a protein comprising an epitope thatbinds to an antibody having the identifying characteristics of humananti-dengue virus antibody m360. The disclosure also provides a nucleicacid molecule that encodes a protein comprising an epitope that binds toan antibody having the identifying characteristics of human anti-denguevirus antibody m3666.

One embodiment is a nucleic acid molecule that encodes a humananti-dengue virus antibody that binds to an envelope protein of denguevirus, wherein the antibody is cross-reactive with dengue virus (DENV)serotype 1 envelope protein, DENV serotype 2 envelope protein, DENVserotype 3 envelope protein, and DENV serotype 4 envelope protein. Inone embodiment, the antibody encoded by the nucleic acid moleculeneutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENVserotype 4. In one embodiment, the antibody encoded by the nucleic acidmolecule binds to a receptor binding domain of the envelope protein. Inone embodiment, the antibody encoded by the nucleic acid molecule bindsto a receptor binding domain of the envelope protein and neutralizesDENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4.In one embodiment, the antibody encoded by the nucleic acid moleculebinds to domain III of the envelope protein. In one embodiment, theantibody encoded by the nucleic acid molecule binds to domain III of theenvelope protein and neutralizes DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4. In one embodiment, the antibody encodedby the nucleic acid molecule binds to each of DENV serotype 1, DENVserotype 2, DENV serotype 3, and DENV serotype 4 envelope proteins witha dissociation constant (K_(D)) of no more than 20 nanomolar (nM)

In one embodiment, the antibody encoded by the nucleic acid molecule isnot isolated from a human subject.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:4, an antibody comprising aminoacid sequence SEQ ID NO:6, an antibody comprising amino acid sequencesSEQ ID NO:4 and SEQ ID NO:6, or an antibody comprising amino acidsequence SEQ ID NO:2. One embodiment is a nucleic acid molecule thatencodes an antibody comprising amino acid sequence SEQ ID NO:4. Oneembodiment is a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:6. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:4 and SEQ ID NO:6. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:2. Oneembodiment is a nucleic acid molecule that encodes an antibodycomprising an amino acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:4,SEQ ID NO:6, and SEQ ID NO:2. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:3, comprising nucleic acid sequence SEQ ID NO:5,comprising nucleic acid sequences SEQ ID NO:3 and SEQ ID NO:5, orcomprising nucleic acid sequence SEQ ID NO:1. One embodiment is anucleic acid molecule comprising nucleic acid sequence SEQ ID NO:3. Oneembodiment is a nucleic acid molecule comprising nucleic acid sequenceSEQ ID NO:5. One embodiment is a nucleic acid molecule comprisingnucleic acid sequences SEQ ID NO:3 and SEQ ID NO:5. One embodiment is anucleic acid molecule comprising nucleic acid sequence SEQ ID NO:1. Oneembodiment is a nucleic acid molecule comprising a nucleic acid sequencethat is at least 50 percent, at least 60 percent, at least 70 percent,at least 80 percent, at least 90 percent, at least 91 percent, at least92 percent, at least 93 percent, at least 94 percent, at least 95percent, at least 96 percent, at least 97 percent, at least 98 percent,or at least 99 percent identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:1.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodyof the embodiments that comprises amino acid sequences SEQ ID NO:4 andSEQ ID NO:6 joined by a peptide linker. One embodiment is a nucleic acidmolecule that encodes an antibody comprising amino acid sequences SEQ IDNO:4 and SEQ ID NO:6 joined by a peptide linker comprising amino acidsequence SEQ ID NO:20. In one embodiment the order of the amino acidsequences from N-terminus to C-terminus is SEQ ID NO:4-SEQ ID NO:20-SEQID NO:6. One embodiment is a nucleic acid molecule that encodes anantibody comprising an amino acid sequence that is at least 50 percent,at least 60 percent, at least 70 percent, at least 80 percent, at least90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequences SEQ ID NO:4 and SEQ ID NO:6 joined bya peptide linker comprising amino acid sequence SEQ ID NO:20. In oneembodiment the order of the amino acid sequences from N-terminus toC-terminus is SEQ ID NO:4-SEQ ID NO:20-SEQ ID NO:6. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that comprises nucleicacid sequences SEQ ID NO:3 and SEQ ID NO:5 joined by a nucleic acidsequence encoding a peptide linker. One embodiment is a nucleic acidmolecule comprising nucleic acid sequences SEQ ID NO:3 and SEQ ID NO:5joined by nucleic acid sequence SEQ ID NO:19. In one embodiment, theorder of the nucleic acid sequences from 5′ to 3′ is SEQ ID NO:3-SEQ IDNO:19-SEQ ID NO:5. One embodiment is a nucleic acid molecule comprisinga nucleic acid sequence that is at least 50 percent, at least 60percent, at least 70 percent, at least 80 percent, at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequences SEQ ID NO:3 and SEQ ID NO:5 joined by nucleicacid sequence SEQ ID NO:19. In one embodiment, the order of the nucleicacid sequences from 5′ to 3′ is SEQ ID NO:3-SEQ ID NO:19-SEQ ID NO:5.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:2. One embodiment is a nucleicacid molecule that encodes an antibody comprising an amino acid sequencethat is at least 50 percent, at least 60 percent, at least 70 percent,at least 80 percent, at least 90 percent, at least 91 percent, at least92 percent, at least 93 percent, at least 94 percent, at least 95percent, at least 96 percent, at least 97 percent, at least 98 percent,or at least 99 percent identical to amino acid sequence SEQ ID NO:2.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:1. One embodiment is a nucleic acid moleculecomprising a nucleic acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequence SEQ ID NO:1. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, and combinations thereof. One embodiment is anucleic acid molecule that encodes a V_(H) having a CDR having an aminoacid sequence selected from the group consisting of SEQ ID NO:8, SEQ IDNO:10, and SEQ ID NO:12, and combinations thereof. One embodiment is anucleic acid molecule that encodes a V_(H), wherein CDR-H1 has aminoacid sequence SEQ ID NO:8, CDR-H2 has amino acid sequence SEQ ID NO:10,and CDR-H3 has amino acid sequence SEQ ID NO:12. One embodiment is anucleic acid molecule that encodes a V_(L), wherein CDR-L1 has aminoacid sequence SEQ ID NO:14, CDR-L2 has amino acid sequence SEQ ID NO:16,and CDR-L3 has amino acid sequence SEQ ID NO:18. Each of these nucleicacid molecules encodes an antibody that retains the ability to bind to aDENV envelope protein and to cross-react with envelope proteins from allfour DENV serotypes. Such an encoded antibody can be a complete Ig orany fragment thereof.

One embodiment is a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and combinationsthereof. One embodiment is a nucleic acid molecule comprising nucleicacid sequences SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. One embodimentis a nucleic acid molecule comprising nucleic acid sequences SEQ IDNO:13, SEQ ID NO:15, and SEQ ID NO:17. Each of these nucleic acidmolecules encodes an antibody that retains the ability to bind to a DENVenvelope protein and to cross-react with envelope proteins from all fourDENV serotypes. Such an encoded antibody can be a complete Ig or anyfragment thereof.

A nucleic acid molecule of the embodiments can include a nucleic acidsequence that encodes an Fc domain of the embodiments. In oneembodiment, such an encoded Fc domain can be joined to a fusion segmentof the embodiments.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a variable domain having the identifying characteristics ofhuman anti-dengue virus antibody m366. One embodiment is a nucleic acidmolecule that encodes a scFv comprising a variable domain having theidentifying characteristics of human anti-dengue virus antibody m366.One embodiment is a first nucleic acid molecule that encodes a heavychain of an antibody comprising a variable domain having the identifyingcharacteristics of human anti-dengue virus antibody m366 and a secondnucleic acid molecule that encodes a light chain of an antibodycomprising a variable domain having the identifying characteristics ofhuman anti-dengue virus antibody m366.

The disclosure provides a nucleic acid molecule that encodes a proteincomprising an epitope that binds to an antibody having the identifyingcharacteristics of human anti-dengue virus antibody m366. Examples ofsuch epitope-containing proteins are provided herein.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:24, an antibody comprisingamino acid sequence SEQ ID NO:26, an antibody comprising amino acidsequences SEQ ID NO:24 and SEQ ID NO:26, or an antibody comprising aminoacid sequence SEQ ID NO:22. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:24.One embodiment is a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:26. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:24 and SEQ ID NO:26. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:22.One embodiment is a nucleic acid molecule that encodes an antibodycomprising an amino acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:24,SEQ ID NO:26, and SEQ ID NO:22. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:23, comprising nucleic acid sequence SEQ ID NO:25,comprising nucleic acid sequences SEQ ID NO:23 and SEQ ID NO:25, orcomprising nucleic acid sequence SEQ ID NO:21. One embodiment is anucleic acid molecule comprising nucleic acid sequence SEQ ID NO:23. Oneembodiment is a nucleic acid molecule comprising nucleic acid sequenceSEQ ID NO:25. One embodiment is a nucleic acid molecule comprisingnucleic acid sequences SEQ ID NO:23 and SEQ ID NO:25. One embodiment isa nucleic acid molecule comprising nucleic acid sequence SEQ ID NO:21.One embodiment is a nucleic acid molecule comprising a nucleic acidsequence that is at least 50 percent, at least 60 percent, at least 70percent, at least 80 percent, at least 90 percent, at least 91 percent,at least 92 percent, at least 93 percent, at least 94 percent, at least95 percent, at least 96 percent, at least 97 percent, at least 98percent, or at least 99 percent identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, andSEQ ID NO:21. Each of these nucleic acid molecules encodes an antibodythat retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anencoded antibody can be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodyof the embodiments that comprises amino acid sequences SEQ ID NO:24 andSEQ ID NO:26 joined by a peptide linker. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:24 and SEQ ID NO:26 joined by a peptide linker comprisingamino acid sequence SEQ ID NO:40. In one embodiment the order of theamino acid sequences from N-terminus to C-terminus is SEQ ID NO:24-SEQID NO:40-SEQ ID NO:26. In one embodiment the order of the amino acidsequences from N-terminus to C-terminus is SEQ ID NO:26-SEQ ID NO:40-SEQID NO:24. One embodiment is a nucleic acid molecule that encodes anantibody comprising an amino acid sequence that is at least 50 percent,at least 60 percent, at least 70 percent, at least 80 percent, at least90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequences SEQ ID NO:24 and SEQ ID NO:26 joinedby a peptide linker comprising amino acid sequence SEQ ID NO:40. In oneembodiment the order of the amino acid sequences from N-terminus toC-terminus is SEQ ID NO:24-SEQ ID NO:40-SEQ ID NO:26. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that comprises nucleicacid sequences SEQ ID NO:23 and SEQ ID NO:25 joined by a nucleic acidsequence encoding a peptide linker. One embodiment is a nucleic acidmolecule comprising nucleic acid sequences SEQ ID NO:23 and SEQ ID NO:25joined by nucleic acid sequence SEQ ID NO:39. In one embodiment, theorder of the nucleic acid sequences from 5′ to 3′ is SEQ ID NO:23-SEQ IDNO:39-SEQ ID NO:25. One embodiment is a nucleic acid molecule comprisinga nucleic acid sequence that is at least 50 percent, at least 60percent, at least 70 percent, at least 80 percent, at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequences SEQ ID NO:23 and SEQ ID NO:25 joined by nucleicacid sequence SEQ ID NO:39. In one embodiment, the order of the nucleicacid sequences from 5′ to 3′ is SEQ ID NO:23-SEQ ID NO:39-SEQ ID NO:25.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:22. One embodiment is a nucleicacid molecule that encodes an antibody comprising an amino acid sequencethat is at least 50 percent, at least 60 percent, at least 70 percent,at least 80 percent, at least 90 percent, at least 91 percent, at least92 percent, at least 93 percent, at least 94 percent, at least 95percent, at least 96 percent, at least 97 percent, at least 98 percent,or at least 99 percent identical to amino acid sequence SEQ ID NO:22.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:21. One embodiment is a nucleic acid moleculecomprising a nucleic acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequence SEQ ID NO:21. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:36, SEQ ID NO:38, and combinations thereof. One embodiment isa nucleic acid molecule that encodes a V_(H) having a CDR having anamino acid sequence selected from the group consisting of SEQ ID NO:28,SEQ ID NO:30, and SEQ ID NO:32, and combinations thereof. One embodimentis a nucleic acid molecule that encodes a V_(H), wherein CDR-H1 hasamino acid sequence SEQ ID NO:28, CDR-H2 has amino acid sequence SEQ IDNO:30, and CDR-H3 has amino acid sequence SEQ ID NO:32. One embodimentis a nucleic acid molecule that encodes a V_(L), wherein CDR-L1 hasamino acid sequence SEQ ID NO:34, CDR-L2 has amino acid sequence SEQ IDNO:36, and CDR-L3 has amino acid sequence SEQ ID NO:38. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

One embodiment is a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, andcombinations thereof. One embodiment is a nucleic acid moleculecomprising nucleic acid sequences SEQ ID NO:27, SEQ ID NO:29 and SEQ IDNO:31. One embodiment is a nucleic acid molecule comprising nucleic acidsequences SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

A nucleic acid molecule of the embodiments can include a nucleic acidsequence that encodes an Fc domain of the embodiments. In oneembodiment, such an encoded Fc domain can be joined to a fusion segmentof the embodiments.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a variable domain having the identifying characteristics ofhuman anti-dengue virus antibody m366.6. One embodiment is a nucleicacid molecule that encodes a scFv comprising a variable domain havingthe identifying characteristics of human anti-dengue virus antibodym366.6. One embodiment is a first nucleic acid molecule that encodes aheavy chain of an antibody comprising a variable domain having theidentifying characteristics of human anti-dengue virus antibody m366.6and a second nucleic acid molecule that encodes a light chain of anantibody comprising a variable domain having the identifyingcharacteristics of human anti-dengue virus antibody m366.6.

The disclosure provides a nucleic acid molecule that encodes a proteincomprising an epitope that binds to an antibody having the identifyingcharacteristics of human anti-dengue virus antibody m366.6. Examples ofsuch epitope-containing proteins are provided herein.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:44, an antibody comprisingamino acid sequence SEQ ID NO:46, an antibody comprising amino acidsequences SEQ ID NO:44 and SEQ ID NO:46, or an antibody comprising aminoacid sequence SEQ ID NO:42. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:44.One embodiment is a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:46. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:44 and SEQ ID NO:46. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:42.One embodiment is a nucleic acid molecule that encodes an antibodycomprising an amino acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:44,SEQ ID NO:46, and SEQ ID NO:42. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:43, comprising nucleic acid sequence SEQ ID NO:45,comprising nucleic acid sequences SEQ ID NO:43 and SEQ ID NO:45, orcomprising nucleic acid sequence SEQ ID NO:41. One embodiment is anucleic acid molecule comprising nucleic acid sequence SEQ ID NO:43. Oneembodiment is a nucleic acid molecule comprising nucleic acid sequenceSEQ ID NO:45. One embodiment is a nucleic acid molecule comprisingnucleic acid sequences SEQ ID NO:43 and SEQ ID NO:45. One embodiment isa nucleic acid molecule comprising nucleic acid sequence SEQ ID NO:41.One embodiment is a nucleic acid molecule comprising a nucleic acidsequence that is at least 50 percent, at least 60 percent, at least 70percent, at least 80 percent, at least 90 percent, at least 91 percent,at least 92 percent, at least 93 percent, at least 94 percent, at least95 percent, at least 96 percent, at least 97 percent, at least 98percent, or at least 99 percent identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:43, SEQ ID NO:45, andSEQ ID NO:41. Each of these nucleic acid molecules encodes an antibodythat retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anencoded antibody can be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodyof the embodiments that comprises amino acid sequences SEQ ID NO:44 andSEQ ID NO:46 joined by a peptide linker. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:44 and SEQ ID NO:46 joined by a peptide linker comprisingamino acid sequence SEQ ID NO:60. In one embodiment the order of theamino acid sequences from N-terminus to C-terminus is SEQ ID NO:44-SEQID NO:60-SEQ ID NO:46. In one embodiment the order of the amino acidsequences from N-terminus to C-terminus is SEQ ID NO:46-SEQ ID NO:60-SEQID NO:44. One embodiment is a nucleic acid molecule that encodes anantibody comprising an amino acid sequence that is at least 50 percent,at least 60 percent, at least 70 percent, at least 80 percent, at least90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequences SEQ ID NO:44 and SEQ ID NO:46 joinedby a peptide linker comprising amino acid sequence SEQ ID NO:60. In oneembodiment the order of the amino acid sequences from N-terminus toC-terminus is SEQ ID NO:44-SEQ ID NO:60-SEQ ID NO:46. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that comprises nucleicacid sequences SEQ ID NO:43 and SEQ ID NO:45 joined by a nucleic acidsequence encoding a peptide linker. One embodiment is a nucleic acidmolecule comprising nucleic acid sequences SEQ ID NO:43 and SEQ ID NO:45joined by nucleic acid sequence SEQ ID NO:59. In one embodiment, theorder of the nucleic acid sequences from 5′ to 3′ is SEQ ID NO:43-SEQ IDNO:59-SEQ ID NO:45. One embodiment is a nucleic acid molecule comprisinga nucleic acid sequence that is at least 50 percent, at least 60percent, at least 70 percent, at least 80 percent, at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequences SEQ ID NO:43 and SEQ ID NO:45 joined by nucleicacid sequence SEQ ID NO:59. In one embodiment, the order of the nucleicacid sequences from 5′ to 3′ is SEQ ID NO:43-SEQ ID NO:59-SEQ ID NO:45.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:42. One embodiment is a nucleicacid molecule that encodes an antibody comprising an amino acid sequencethat is at least 50 percent, at least 60 percent, at least 70 percent,at least 80 percent, at least 90 percent, at least 91 percent, at least92 percent, at least 93 percent, at least 94 percent, at least 95percent, at least 96 percent, at least 97 percent, at least 98 percent,or at least 99 percent identical to amino acid sequence SEQ ID NO:42.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:41. One embodiment is a nucleic acid moleculecomprising a nucleic acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequence SEQ ID NO:41. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,SEQ ID NO:56, SEQ ID NO:58, and combinations thereof. One embodiment isa nucleic acid molecule that encodes a V_(H) having a CDR having anamino acid sequence selected from the group consisting of SEQ ID NO:48,SEQ ID NO:50, and SEQ ID NO:52, and combinations thereof. One embodimentis a nucleic acid molecule that encodes a V_(H), wherein CDR-H1 hasamino acid sequence SEQ ID NO:48, CDR-H2 has amino acid sequence SEQ IDNO:50, and CDR-H3 has amino acid sequence SEQ ID NO:52. One embodimentis a nucleic acid molecule that encodes a V_(L), wherein CDR-L1 hasamino acid sequence SEQ ID NO:54, CDR-L2 has amino acid sequence SEQ IDNO:56, and CDR-L3 has amino acid sequence SEQ ID NO:58. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

One embodiment is a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, andcombinations thereof. One embodiment is a nucleic acid moleculecomprising nucleic acid sequences SEQ ID NO:47, SEQ ID NO:49 and SEQ IDNO:51. One embodiment is a nucleic acid molecule comprising nucleic acidsequences SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

A nucleic acid molecule of the embodiments can include a nucleic acidsequence that encodes an Fc domain of the embodiments. In oneembodiment, such an encoded Fc domain can be joined to a fusion segmentof the embodiments.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a variable domain having the identifying characteristics ofhuman anti-dengue virus antibody m360.6. One embodiment is a nucleicacid molecule that encodes a scFv comprising a variable domain havingthe identifying characteristics of human anti-dengue virus antibodym360.6. One embodiment is a first nucleic acid molecule that encodes aheavy chain of an antibody comprising a variable domain having theidentifying characteristics of human anti-dengue virus antibody m360.6and a second nucleic acid molecule that encodes a light chain of anantibody comprising a variable domain having the identifyingcharacteristics of human anti-dengue virus antibody m360.6.

The disclosure provides a nucleic acid molecule that encodes a proteincomprising an epitope that binds to an antibody having the identifyingcharacteristics of human anti-dengue virus antibody m360.6. Examples ofsuch epitope-containing proteins are provided herein.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:64, an antibody comprisingamino acid sequence SEQ ID NO:66, an antibody comprising amino acidsequences SEQ ID NO:64 and SEQ ID NO:66, or an antibody comprising aminoacid sequence SEQ ID NO:62. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:64.One embodiment is a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:66. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:64 and SEQ ID NO:66. One embodiment is a nucleic acid moleculethat encodes an antibody comprising amino acid sequence SEQ ID NO:62.One embodiment is a nucleic acid molecule that encodes an antibodycomprising an amino acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:64,SEQ ID NO:66, and SEQ ID NO:62. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:63, comprising nucleic acid sequence SEQ ID NO:65,comprising nucleic acid sequences SEQ ID NO:63 and SEQ ID NO:65, orcomprising nucleic acid sequence SEQ ID NO:61. One embodiment is anucleic acid molecule comprising nucleic acid sequence SEQ ID NO:63. Oneembodiment is a nucleic acid molecule comprising nucleic acid sequenceSEQ ID NO:65. One embodiment is a nucleic acid molecule comprisingnucleic acid sequences SEQ ID NO:63 and SEQ ID NO:65. One embodiment isa nucleic acid molecule comprising nucleic acid sequence SEQ ID NO:61.One embodiment is a nucleic acid molecule comprising a nucleic acidsequence that is at least 50 percent, at least 60 percent, at least 70percent, at least 80 percent, at least 90 percent, at least 91 percent,at least 92 percent, at least 93 percent, at least 94 percent, at least95 percent, at least 96 percent, at least 97 percent, at least 98percent, or at least 99 percent identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:63, SEQ ID NO:65, andSEQ ID NO:61. Each of these nucleic acid molecules encodes an antibodythat retains the ability to bind to a DENV envelope protein and tocross-react with envelope proteins from all four DENV serotypes. Such anencoded antibody can be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodyof the embodiments that comprises amino acid sequences SEQ ID NO:64 andSEQ ID NO:66 joined by a peptide linker. One embodiment is a nucleicacid molecule that encodes an antibody comprising amino acid sequencesSEQ ID NO:64 and SEQ ID NO:66 joined by a peptide linker comprisingamino acid sequence SEQ ID NO:80. In one embodiment the order of theamino acid sequences from N-terminus to C-terminus is SEQ ID NO:64-SEQID NO:80-SEQ ID NO:66. In one embodiment the order of the amino acidsequences from N-terminus to C-terminus is SEQ ID NO:66-SEQ ID NO:80-SEQID NO:64. One embodiment is a nucleic acid molecule that encodes anantibody comprising an amino acid sequence that is at least 50 percent,at least 60 percent, at least 70 percent, at least 80 percent, at least90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequences SEQ ID NO:64 and SEQ ID NO:66 joinedby a peptide linker comprising amino acid sequence SEQ ID NO:80. In oneembodiment the order of the amino acid sequences from N-terminus toC-terminus is SEQ ID NO:64-SEQ ID NO:80-SEQ ID NO:66. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that comprises nucleicacid sequences SEQ ID NO:63 and SEQ ID NO:65 joined by a nucleic acidsequence encoding a peptide linker. One embodiment is a nucleic acidmolecule comprising nucleic acid sequences SEQ ID NO:63 and SEQ ID NO:65joined by nucleic acid sequence SEQ ID NO:79. In one embodiment, theorder of the nucleic acid sequences from 5′ to 3′ is SEQ ID NO:63-SEQ IDNO:79-SEQ ID NO:65. One embodiment is a nucleic acid molecule comprisinga nucleic acid sequence that is at least 50 percent, at least 60percent, at least 70 percent, at least 80 percent, at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent, at least 96 percent, at least 97percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequences SEQ ID NO:63 and SEQ ID NO:65 joined by nucleicacid sequence SEQ ID NO:79. In one embodiment, the order of the nucleicacid sequences from 5′ to 3′ is SEQ ID NO:63-SEQ ID NO:79-SEQ ID NO:65.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising amino acid sequence SEQ ID NO:62. One embodiment is a nucleicacid molecule that encodes an antibody comprising an amino acid sequencethat is at least 50 percent, at least 60 percent, at least 70 percent,at least 80 percent, at least 90 percent, at least 91 percent, at least92 percent, at least 93 percent, at least 94 percent, at least 95percent, at least 96 percent, at least 97 percent, at least 98 percent,or at least 99 percent identical to amino acid sequence SEQ ID NO:62.Each of these nucleic acid molecules encodes an antibody that retainsthe ability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes. Such an encoded antibodycan be a complete Ig or any fragment thereof.

The disclosure provides a nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:61. One embodiment is a nucleic acid moleculecomprising a nucleic acid sequence that is at least 50 percent, at least60 percent, at least 70 percent, at least 80 percent, at least 90percent, at least 91 percent, at least 92 percent, at least 93 percent,at least 94 percent, at least 95 percent, at least 96 percent, at least97 percent, at least 98 percent, or at least 99 percent identical tonucleic acid sequence SEQ ID NO:61. Each of these nucleic acid moleculesencodes an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes. Such an encoded antibody can be a complete Ig or any fragmentthereof.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a CDR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,SEQ ID NO:76, SEQ ID NO:78, and combinations thereof. One embodiment isa nucleic acid molecule that encodes a V_(H) having a CDR having anamino acid sequence selected from the group consisting of SEQ ID NO:68,SEQ ID NO:70, and SEQ ID NO:72, and combinations thereof. One embodimentis a nucleic acid molecule that encodes a V_(H), wherein CDR-H1 hasamino acid sequence SEQ ID NO:68, CDR-H2 has amino acid sequence SEQ IDNO:70, and CDR-H3 has amino acid sequence SEQ ID NO:72. One embodimentis a nucleic acid molecule that encodes a V_(L), wherein CDR-L1 hasamino acid sequence SEQ ID NO:74, CDR-L2 has amino acid sequence SEQ IDNO:76, and CDR-L3 has amino acid sequence SEQ ID NO:78. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

One embodiment is a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, andcombinations thereof. One embodiment is a nucleic acid moleculecomprising nucleic acid sequences SEQ ID NO:67, SEQ ID NO:69 and SEQ IDNO:71. One embodiment is a nucleic acid molecule comprising nucleic acidsequences SEQ ID NO:73, SEQ ID NO:75, and SEQ ID NO:77. Each of thesenucleic acid molecules encodes an antibody that retains the ability tobind to a DENV envelope protein and to cross-react with envelopeproteins from all four DENV serotypes. Such an encoded antibody can be acomplete Ig or any fragment thereof.

A nucleic acid molecule of the embodiments can include a nucleic acidsequence that encodes an Fc domain of the embodiments. In oneembodiment, such an encoded Fc domain can be joined to a fusion segmentof the embodiments.

The disclosure provides a nucleic acid molecule that encodes an antibodycomprising a variable domain having the identifying characteristics ofhuman anti-dengue virus antibody m360. One embodiment is a nucleic acidmolecule that encodes a scFv comprising a variable domain having theidentifying characteristics of human anti-dengue virus antibody m360.One embodiment is a first nucleic acid molecule that encodes a heavychain of an antibody comprising a variable domain having the identifyingcharacteristics of human anti-dengue virus antibody m360 and a secondnucleic acid molecule that encodes a light chain of an antibodycomprising a variable domain having the identifying characteristics ofhuman anti-dengue virus antibody m360.

The disclosure provides a nucleic acid molecule that encodes a proteincomprising an epitope that binds to an antibody having the identifyingcharacteristics of human anti-dengue virus antibody m360. Examples ofsuch epitope-containing proteins are provided herein.

The disclosure provides a nucleic acid molecule that encodes abispecific human anti-dengue virus antibody of the embodiments. Such abispecific antibody binds to domain III of the envelope protein ofdengue virus and is cross-reactive with domain III of dengue virus(DENV) serotype 1 envelope protein, domain III of DENV serotype 2envelope protein, domain III of DENV serotype 3 envelope protein, anddomain III of DENV serotype 4 envelope protein. Such a bispecificantibody neutralizes DENV serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4.

One embodiment is a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody that comprises two monospecific humananti-dengue virus antibodies of the embodiments. In one embodiment, suchmonospecific antibodies are the same human anti-dengue virus antibody.In one embodiment, such monospecific antibodies are different humananti-dengue virus antibodies that recognize different DENV virusepitopes. One embodiment is a nucleic acid molecule that encodes abispecific antibody comprising a monospecific human anti-dengue virusantibody of the embodiments and an antibody against another target.

The disclosure provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that neutralizes each of DENVserotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4 at anIC₅₀ of less than 25 micrograms per ml (μg/ml), less than 20 μg/ml, lessthan 15 μg/ml, less than 10 μg/ml, less than 5 μg/ml, less than 1 μg/ml,less than 0.5 μg/ml, less than 0.1 μg/ml, less than 0.05 μg/ml, or lessthan 0.01 μg/ml. In one embodiment, such a bispecific anti-dengue virusantibody neutralizes each of DENV serotype 1, DENV serotype 2, DENVserotype 3, and DENV serotype 4 at an IC₅₀ of less than 1 μg/ml. In oneembodiment, such a bispecific anti-dengue virus antibody neutralizes twoof the DENV serotypes at an IC₅₀ of less than 1μg/ml and two of the DENVserotypes at an IC₅₀ of less than 0.1μg/ml. A DENV RVP assay as setforth in the Examples can be used to determine neutralization IC₅₀values.

The disclosure provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that comprises a human anti-denguevirus antibody that binds to each of DENV serotype 1, DENV serotype 2,DENV serotype 3, and DENV serotype 4 envelope proteins with adissociation constant (K_(D)) of no more than 50 nanomolar (nM), no morethan 40 nM, no more than 35 nM, no more than 30 nM, no more than 25 nM,no more than 20 nM, no more than 15 nM. no more than 10 nM, no more than5 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.1 nM, nomore than 0.05 nM, no more than 0.01 nM, no more than 5 pM or no morethan 1 pM. In one embodiment, such a bispecific antibody comprises ahuman anti-dengue virus antibody that binds to each of DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4 envelope proteinswith a K_(D) of no more than 40 nM. In one embodiment such a bispecificantibody comprises a human anti-dengue virus antibody that binds to eachof DENV serotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype4 envelope proteins with a K_(D) of no more than 20 nM. In oneembodiment, such a bispecific antibody comprises a human anti-denguevirus antibody that binds to each of DENV serotype 1, DENV serotype 2,DENV serotype 3, and DENV serotype 4 envelope proteins with a K_(D) ofno more than 1 nM. In one embodiment, such a bispecific antibodycomprises a human anti-dengue virus antibody that binds to three DENVserotypes with a K_(D) of no more than 0.5 nM and binds the fourth DENVserotype with a K_(D) of no more than 40 nM.

One embodiment is a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody that comprises a V_(H) chain comprising aminoacid sequence SEQ ID NO:4 and a V_(L) chain comprising amino acidsequence SEQ ID NO:6. One embodiment is a nucleic acid molecule thatencodes a bispecific anti-dengue virus antibody that comprises a V_(H)chain comprising amino acid sequence SEQ ID NO:24 and a V_(L) chaincomprising amino acid sequence SEQ ID NO:26. One embodiment is a nucleicacid molecule that encodes a bispecific anti-dengue virus antibody thatcomprises a V_(H) chain comprising amino acid sequence SEQ ID NO:44 anda V_(L) chain comprising amino acid sequence SEQ ID NO:46. Oneembodiment is a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody that comprises amino acid sequence SEQ IDNO:2. One embodiment is a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that comprises amino acid sequenceSEQ ID NO:22. One embodiment is a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that comprises amino acid sequenceSEQ ID NO:42.

The disclosure provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that comprises: an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:4 anda V_(L) chain comprising amino acid sequence SEQ ID NO:6; and anantibody comprising a V_(H) chain comprising amino acid sequence SEQ IDNO:24 and a V_(L) chain comprising amino acid sequence SEQ ID NO:26. Thedisclosure also provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that comprises: an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:4 anda V_(L) chain comprising amino acid sequence SEQ ID NO:6; and anantibody comprising a V_(H) chain comprising amino acid sequence SEQ IDNO:44 and a V_(L) chain comprising amino acid sequence SEQ ID NO:46. Thedisclosure also provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody that comprises: an antibodycomprising a V_(H) chain comprising amino acid sequence SEQ ID NO:24 anda V_(L) chain comprising amino acid sequence SEQ ID NO:26; and anantibody comprising a V_(H) chain comprising amino acid sequence SEQ IDNO:44 and a V_(L) chain comprising amino acid sequence SEQ ID NO:46. Oneembodiment is a nucleic acid molecule that encodes a bispecific antibodycomprising an antibody comprising amino acid sequence SEQ ID NO:2 and anantibody comprising amino acid sequence SEQ ID NO:22. One embodiment isa nucleic acid molecule that encodes a bispecific antibody comprising anantibody comprising amino acid sequence SEQ ID NO:2 and an antibodycomprising amino acid sequence SEQ ID NO:42. One embodiment is a nucleicacid molecule that encodes a bispecific antibody comprising an antibodycomprising amino acid sequence SEQ ID NO:22 and an antibody comprisingamino acid sequence SEQ ID NO:42.

The disclosure also provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody comprising an Fc domain. In oneembodiment, the Fc domain is glycosylated. In one embodiment, the Fcdomain is non-glycosylated. In one embodiment, an Fc domain has modifiedaffinity to one or more Fc receptors. In one embodiment, the Fc domainexhibits reduced binding to an Fc gamma receptor. In one embodiment, theFc domain is flanked by linkers. One embodiment is a nucleic acidsequence that encodes a bispecific anti-dengue antibody having an Fcdomain comprising amino acid sequence SEQ ID NO:102. One embodiment is anucleic acid sequence that encodes a bispecific anti-dengue antibodyhaving an Fc domain comprising amino acid sequence SEQ ID NO:104. Oneembodiment is a nucleic acid molecule that encodes a bispecificanti-dengue antibody having an Fc domain comprising amino acid sequenceSEQ ID NO:92. One embodiment is a nucleic acid molecule that encodes abispecific anti-dengue antibody having an Fc domain comprising aminoacid sequence SEQ ID NO:94. One embodiment is a nucleic acid moleculethat comprises nucleic acid sequence SEQ ID NO:101. One embodiment is anucleic acid molecule that comprises nucleic acid sequence SEQ IDNO:103. One embodiment is a nucleic acid molecule that comprises nucleicacid sequence SEQ ID NO:91. One embodiment is a nucleic acid moleculethat comprises nucleic acid sequence SEQ ID NO:93.

The disclosure provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody comprising amino acid sequencesSEQ ID NO:2, SEQ ID NO:102, and SEQ ID NO:22. The disclosure provides anucleic acid molecule that encodes a bispecific anti-dengue virusantibody comprising amino acid sequences SEQ ID NO:2, SEQ ID NO:104, andSEQ ID NO:22. The disclosure provides a nucleic acid molecule thatencodes a bispecific anti-dengue virus antibody comprising amino acidsequences SEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:22. The disclosurealso provides a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody comprising amino acid sequences SEQ ID NO:2,SEQ ID NO:94, and SEQ ID NO:22. In one embodiment, the encodedbispecific antibody has an amino acid sequence in the order SEQ ID:2-SEQID NO:94-SEQ ID NO:22. In one embodiment, the encoded bispecificantibody has an amino acid sequence in the order SEQ ID:22-SEQ IDNO:94-SEQ ID NO:2. One embodiment is a nucleic acid molecule comprisingSEQ ID NO:1, SEQ ID NO:93, and SEQ ID NO:21.

The disclosure provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody comprising amino acid sequencesSEQ ID NO:2, SEQ ID NO:102, and SEQ ID NO:42. The disclosure provides anucleic acid molecule that encodes a bispecific anti-dengue virusantibody comprising amino acid sequences SEQ ID NO:2, SEQ ID NO:104, andSEQ ID NO:42. The disclosure provides a nucleic acid molecule thatencodes a bispecific anti-dengue virus antibody comprising amino acidsequences SEQ ID NO:2, SEQ ID NO:92, and SEQ ID NO:42. The disclosurealso provides a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody comprising amino acid sequences SEQ ID NO:2,SEQ ID NO:94, and SEQ ID NO:42. In one embodiment, the encodedbispecific antibody has an amino acid sequence in the order SEQ ID:2-SEQID NO:94-SEQ ID NO:42. In one embodiment, the encoded bispecificantibody has an amino acid sequence in the order SEQ ID:42-SEQ IDNO:94-SEQ ID NO:2. One embodiment is a nucleic acid molecule comprisingSEQ ID NO:1, SEQ ID NO:93, and SEQ ID NO:41.

The disclosure provides a nucleic acid molecule that encodes abispecific anti-dengue virus antibody comprising amino acid sequencesSEQ ID NO:22, SEQ ID NO:102, and SEQ ID NO:42. The disclosure provides anucleic acid molecule that encodes a bispecific anti-dengue virusantibody comprising amino acid sequences SEQ ID NO:22, SEQ ID NO:104,and SEQ ID NO:42. The disclosure provides a nucleic acid molecule thatencodes a bispecific anti-dengue virus antibody comprising amino acidsequences SEQ ID NO:22, SEQ ID NO:92, and SEQ ID NO:42. The disclosurealso provides a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody comprising amino acid sequences SEQ ID NO:22,SEQ ID NO:94, and SEQ ID NO:42. In one embodiment, the encodedbispecific antibody has an amino acid sequence in the order SEQID:22-SEQ ID NO:94-SEQ ID NO:42. In one embodiment, the encodedbispecific antibody has an amino acid sequence in the order SEQID:42-SEQ ID NO:94-SEQ ID NO:22. One embodiment is a nucleic acidmolecule comprising SEQ ID NO:21, SEQ ID NO:93, and SEQ ID NO:41. Oneembodiment is a nucleic acid molecule that has a nucleic acid sequencein the order of SEQ ID NO:41-SEQ ID NO:93-SEQ ID NO:21. One embodimentis a nucleic acid molecule that has a nucleic acid sequence in the orderof SEQ ID NO:21-SEQ ID NO:93-SEQ ID NO:41.

One embodiment is a nucleic acid molecule that encodes a bispecificanti-dengue virus antibody comprising amino acid sequence SEQ ID NO:96.One embodiment is a nucleic acid molecule comprising SEQ ID NO:95.

One embodiment is a nucleic acid molecule that encodes a bispecificantibody comprising an amino acid sequence that is at least 50 percent,at least 60 percent, at least 70 percent, at least 80 percent, at least90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, or at least 99 percentidentical to amino acid sequence SEQ ID NO:96. One embodiment is anucleic acid molecule that encodes a bispecific antibody comprisingamino acid sequence SEQ ID NO:96. One embodiment is a nucleic acidmolecule comprising a nucleic acid sequence that is at least 50 percent,at least 60 percent, at least 70 percent, at least 80 percent, at least90 percent, at least 91 percent, at least 92 percent, at least 93percent, at least 94 percent, at least 95 percent, at least 96 percent,at least 97 percent, at least 98 percent, at least 99 percent, or 100percent identical to nucleic acid sequence SEQ ID NO:95. Each of thesenucleic acid molecules encodes a bispecific antibody that retains theability to bind to a DENV envelope protein and to cross-react withenvelope proteins from all four DENV serotypes.

The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibody m366.The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibody m366.6.The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibody m360.6.The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibody m360.The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibodies m366and m366.6. The disclosure provides a nucleic acid molecule that encodesa bispecific antibody comprising human anti-dengue virus antibodies m366and m360.6. The disclosure provides a nucleic acid molecule that encodesa bispecific antibody comprising human anti-dengue virus antibodies m366and m360. The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibodies m366.6and m360.6. The disclosure provides a nucleic acid molecule that encodesa bispecific antibody comprising human anti-dengue virus antibodiesm366.6 and m360. The disclosure provides a nucleic acid molecule thatencodes a bispecific antibody comprising human anti-dengue virusantibodies m360.6 and m360.

The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibody m366 andan Fc domain, such as an Fc domain comprising amino acid sequence SEQ IDNO:92 or SEQ ID NO:94, such as an Fc domain comprising amino acidsequence SEQ ID NO:94. The disclosure provides a nucleic acid moleculethat encodes a bispecific antibody comprising human anti-dengue virusantibody m366.6 and an Fc domain, such as an Fc domain comprising aminoacid sequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domaincomprising amino acid sequence SEQ ID NO:94. The disclosure provides anucleic acid molecule that encodes a bispecific antibody comprisinghuman anti-dengue virus antibody m360.6 and an Fc domain, such as an Fcdomain comprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, suchas an Fc domain comprising amino acid sequence SEQ ID NO:94. Thedisclosure provides a nucleic acid molecule that encodes a bispecificantibody comprising human anti-dengue virus antibody m360 and an Fcdomain, such as an Fc domain comprising amino acid sequence SEQ ID NO:92or SEQ ID NO:94, such as an Fc domain comprising amino acid sequence SEQID NO:94.

The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibodies m366and m366.6 and an Fc domain, such as an Fc domain comprising amino acidsequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain comprisingamino acid sequence SEQ ID NO:94. The disclosure provides a nucleic acidmolecule that encodes a bispecific antibody comprising human anti-denguevirus antibodies m366 and m360.6 and an Fc domain, such as an Fc domaincomprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as anFc domain comprising amino acid sequence SEQ ID NO:94. The disclosureprovides a nucleic acid molecule that encodes a bispecific antibodycomprising human anti-dengue virus antibodies m366 and m360 and an Fcdomain, such as an Fc domain comprising amino acid sequence SEQ ID NO:92or SEQ ID NO:94, such as an Fc domain comprising amino acid sequence SEQID NO:94. The disclosure provides a nucleic acid molecule that encodes abispecific antibody comprising human anti-dengue virus antibodies m366.6and m360.6 and an Fc domain, such as an Fc domain comprising amino acidsequence SEQ ID NO:92 or SEQ ID NO:94, such as an Fc domain comprisingamino acid sequence SEQ ID NO:94. The disclosure provides a nucleic acidmolecule that encodes a bispecific antibody comprising human anti-denguevirus antibodies m366.6 and m360 and an Fc domain, such as an Fc domaincomprising amino acid sequence SEQ ID NO:92 or SEQ ID NO:94, such as anFc domain comprising amino acid sequence SEQ ID NO:94. The disclosureprovides a nucleic acid molecule that encodes a bispecific antibodycomprising human anti-dengue virus antibodies m360.6 and m360 and an Fcdomain, such as an Fc domain comprising amino acid sequence SEQ ID NO:92or SEQ ID NO:94, such as an Fc domain comprising amino acid sequence SEQID NO:94.

The disclosure provides a nucleic acid molecule that encodes abispecific antibody having the identifying characteristics of bispecificanti-dengue virus antibody m3666. The disclosure also provides a nucleicacid molecule that encodes bispecific anti-dengue virus antibody m3666.

The disclosure provides a nucleic acid molecule that encodes a proteincomprising an epitope that binds to an antibody having the identifyingcharacteristics of bispecific anti-dengue virus antibody m3666. Examplesof such epitope-containing proteins are provided herein.

The disclosure provides a method to produce a human anti-dengue virusantibody of the embodiments. The method comprises: (a) screening a yeastdisplay human antibody library for a human antibody cross-reactive withdengue virus (DENV) serotype 1, DENV serotype 2, DENV serotype 3, andDENV serotype 4 in the presence of a non-neutralizing DENV envelopedomain III protein; and (b) isolating a clone expressing the antibodycross-reactive with DENV serotype 1, DENV serotype 2, DENV serotype 3,and DENV serotype 4. In one embodiment, the non-neutralizing DENVenvelope domain III protein has a mutation at a residue corresponding toamino acid residue 20 of amino acid sequence SEQ ID NO:82. In oneembodiment, the mutation is a K310E point mutation, wherein the mutationis at amino acid residue 310 of amino acid sequence SEQ ID NO:81.

The disclosure also provides a method to produce an human anti-denguevirus antibody of the embodiments, which comprises: (a) culturing arecombinant cell encoding the antibody; and (b) recovering the antibody.

Nucleic acid molecules of the embodiments can be produced using a numberof methods known to those skilled in the art; see, for example, SambrookJ et al., 2001, Molecular Cloning: a Laboratory Manual, 3^(rd) edition,Cold Spring Harbor Laboratory Press, and Ausubel F et al., 1994, CurrentProtocols in Molecular Biology, John Wiley & Sons. For example, nucleicacid molecules can be modified using a variety of techniques including,but not limited to, classic mutagenesis techniques and recombinant DNAtechniques, such as site-directed mutagenesis, chemical treatment of anucleic acid molecule to induce mutations, restriction enzyme cleavageof a nucleic acid fragment, ligation of nucleic acid fragments,polymerase chain reaction (PCR) amplification and/or mutagenesis ofselected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecules of the embodiments can be selected from a mixture of modifiednucleic acids by screening for the function of the protein encoded bythe nucleic acid (e.g., the ability of such a nucleic acid molecule toencode an antibody that retains the ability to bind to a DENV envelopeprotein and to cross-react with envelope proteins from all four DENVserotypes).

Methods useful for producing antibodies of the embodiments are known tothose skilled in the art and are also exemplified in the Examples. Suchantibodies can be produced synthetically using techniques known to thoseskilled in the art. Such antibodies can be produced using recombinanttechniques known to those skilled in the art.

The disclosure provides a recombinant vector that comprises a nucleicacid encoding an antibody of the embodiments joined to a vector. Such avector can be a plasmid vector, a viral vector, or other vector. Such avector can be DNA, RNA, or a derivative of DNA or RNA

The disclosure provides a recombinant molecule that comprises a nucleicacid encoding an antibody of the embodiments operatively linked to atleast one transcription control sequence capable of effecting expressionof the nucleic acid molecule in a recombinant cell. A recombinant cellis a host cell that is transformed with a recombinant molecule of theembodiments; i.e., a recombinant cell comprises a recombinant molecule.A recombinant molecule can comprise one or more nucleic acid moleculesencoding an antibody of the embodiments operatively linked to one ormore transcription control sequences. As used herein, the term“operatively linked” refers to the nucleic acid molecule being joined tothe transcription control sequence in a manner to enable expression ofthe nucleic acid molecule in the recombinant cell. A recombinantmolecule can also contain other regulatory control sequences known tothose skilled in the art. Examples of regulatory control sequencesinclude, but are not limited to, promoters, enhancers, repressors,ribosome binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and microRNA binding sites. Arecombinant cell can comprise one or more recombinant molecules. Hostcells to transform can be selected based on their ability to effectexpression of a nucleic acid molecule of the embodiments. Host cells canalso be selected that effect post-translational modifications. Methodsto select, produce and use recombinant vectors, recombinant molecules,and recombinant cells of the embodiments are known to those skilled inthe art. Antibodies of the embodiments can be produced by culturingrecombinant cells of the embodiments. Methods to effect such productionand recovery of such antibodies are known to those skilled in the art,see for example Sambrook J et al., ibid, and Ausubel, F et al., ibid.

The disclosure provides a method to produce a protein that binds to anantibody having the identifying characteristics of a human anti-denguevirus antibody selected from the group consisting of antibody m366,antibody m366.6, antibody m360.6, antibody m360, or antibody m3666. Themethod comprises: (a) culturing a recombinant cell encoding anepitope-containing protein of the embodiments; and (b) recovering theprotein. The disclosure also provides a recombinant vector comprising anucleic acid molecule that encodes a protein that binds to an antibodyhaving the identifying characteristics of a human anti-dengue virusantibody selected from the group consisting of antibody m366, antibodym366.6, antibody m360.6, antibody m360, or antibody m3666. Oneembodiment is a recombinant molecule comprising a nucleic acid moleculethat encodes a protein that binds to an antibody having the identifyingcharacteristics of a human anti-dengue virus antibody selected from thegroup consisting of antibody m366, antibody m366.6, antibody m360.6,antibody m360, or antibody m3666. The disclosure also provides arecombinant cell comprising a recombinant molecule comprising a nucleicacid molecule that encodes a protein that binds to an antibody havingthe identifying characteristics of a human anti-dengue virus antibodyselected from the group consisting of antibody m366, antibody m366.6,antibody m360.6, antibody m360, or antibody m3666. Epitope-containingproteins of the embodiments can be produced using techniques similar tothose described for antibodies of the embodiments, as known by thoseskilled in the art.

The disclosure provides a method to produce a protein comprising anepitope that elicits production of an antibody having the identifyingcharacteristics of a human anti-dengue virus antibody selected from thegroup consisting of antibody m366, antibody m366.6, antibody m360.6,antibody m360, or antibody m3666, which comprises the steps of: (a)identifying an epitope for the human anti-dengue virus antibody on aDENV envelope protein; (b) engineering the epitope to increase bindingto the corresponding germline antibody using mutagenesis and selectiontechniques; (c) constructing a nucleic acid molecule that expresses theengineered epitope; (d) culturing a recombinant cell expressing thenucleic acid molecule; and (e) recovering epitope. Methods to effecteach of the steps are known to those skilled in the art.

Compositions and Methods of Use to Protect a Subject From DENV Infection

The disclosure provides a composition comprising a human anti-denguevirus antibody that binds to an envelope protein of dengue virus,wherein the antibody is cross-reactive with DENV serotype 1 envelopeprotein, DENV serotype 2 envelope protein, DENV serotype 3 envelopeprotein, and DENV serotype 4 envelope protein. In one embodiment, theenvelope protein is domain III of the envelope protein. Such acomposition can also include a carrier. One embodiment is apharmaceutical composition comprising a human anti-dengue virus antibodythat binds to an envelope protein of dengue virus, wherein the antibodyis cross-reactive with DENV serotype 1 envelope protein, DENV serotype 2envelope protein, DENV serotype 3 envelope protein, and DENV serotype 4envelope protein. In one embodiment, the envelope protein is domain IIIof the envelope protein. Such a pharmaceutical composition can alsoinclude a pharmaceutically acceptable carrier. Carriers andpharmaceutically acceptable carriers are known to those skilled in theart. Examples include, but are not limited to, aqueous solutions, suchas a buffer, e.g., a physiologically compatible buffer.

A composition, such as a pharmaceutical composition, of the embodimentsis conveniently formulated in a form suitable for administration to asubject. Techniques to formulate such compositions are known to thoseskilled in the art.

The disclosure provides a method to protect a subject from dengue virusinfection. Such a method comprises administering to the subject a humananti-dengue virus antibody that binds to an envelope protein of denguevirus, wherein the antibody is cross-reactive with DENV serotype 1envelope protein, DENV serotype 2 envelope protein, DENV serotype 3envelope protein, and DENV serotype 4 envelope protein. In oneembodiment, the envelope protein is domain III of the envelope protein.In one embodiment, a pharmaceutical composition comprising such anantibody is administered. As used herein, the ability of an antibody ofthe embodiments to protect a subject from dengue virus infection refersto the ability of the antibody to treat, prevent, reduce, or amelioratethe symptoms of dengue virus infection. In one embodiment, an antibodyof the embodiments prevents dengue virus infection in a subject. In oneembodiment, an antibody of the embodiments treats dengue virus infectionin a subject. In one embodiment, an antibody of the embodiments does notenhance dengue virus infection when administered to a subject. In oneembodiment, an antibody of the embodiments prevents symptoms of denguevirus infection in a subject. In one embodiment, an antibody of theembodiments treats symptoms of dengue virus infection in a subject. Inone embodiment, an antibody of the embodiments prevents symptoms ofdengue virus infection from worsening in a subject. In one embodiment,an antibody of the embodiments ameliorates symptoms of dengue virusinfection in a subject. In one embodiment, an antibody of theembodiments reduces symptoms of dengue virus infection in a subject.Symptoms of dengue virus infection are known to those skilled in theart. Examples of symptoms include, but are not limited to, high fever,severe headache, severe eye pain, joint pain, muscle pain, skin rash,bleeding (e.g., nose or gum bleed, petechiae, easy bruising), low whitecell count, low platelet count, blood plasma leakage, low blood pressuredue to dengue shock syndrome, and dengue hemorrhagic fever. In somecases, high fever is combined with one or more additional symptoms.Methods to identify and measure such symptoms are known to those skilledin the art.

As used herein, a subject is any animal that is susceptible to denguevirus infection. Subjects include humans and non-human primates. In oneembodiment, a subject is a human. In one embodiment, a subject is anon-human primate.

An antibody of the embodiments can be administered in a variety of ways,such as by oral, intranasal, intraocular, conjunctival, intravenous,intraperitoneal, intramuscular, subcutaneous, intradermal, transdermal,topical, aerosol, and rectal administration routes.

The dose of antibodies disclosed herein to be administered to a subjectto be effective (i.e., to protect a subject from dengue virus infection)will depend on the subject's condition, manner of administration, andjudgment of the prescribing physician. One skilled in the art candetermine the dose as well as a dosing regimen that defines the numberand scheduling of doses required.

An antibody of the disclosure can be administered alone or incombination with one or more other antibodies. Examples of otherantibodies include, but are limited to, other anti-dengue antibodies aswell as antibodies against other diseases. Examples of other anti-dengueantibodies include one or more antibodies of the disclosure. Oneembodiment is a cocktail of antibodies, such as a cocktail of humananti-dengue virus antibodies of the disclosure. An antibody of thedisclosure can also be administered in combination with one or moreother therapeutic agents. For example, an antibody of the disclosure canbe administered in addition to an epitope-containing protein of thedisclosure. The antibody and protein can be administered together or oneprior to the other separated by a time known to those skilled in theart.

The disclosure provides a treatment for dengue virus infectioncomprising a human anti-dengue virus antibody that binds to an envelopeprotein of dengue virus, wherein the antibody is cross-reactive withDENV serotype 1 envelope protein, DENV serotype 2 envelope protein, DENVserotype 3 envelope protein, and DENV serotype 4 envelope protein and apharmaceutically acceptable carrier. In one embodiment, the envelopeprotein is domain III of the envelope protein. The disclosure provides apreventative composition against dengue virus infection comprising ahuman anti-dengue virus antibody that binds to an envelope protein ofdengue virus, wherein the antibody is cross-reactive with DENV serotype1 envelope protein, DENV serotype 2 envelope protein, DENV serotype 3envelope protein, and DENV serotype 4 envelope protein and apharmaceutically acceptable carrier.

In one embodiment, the envelope protein is domain III of the envelopeprotein.

The disclosure provides for a human anti-dengue virus antibody thatbinds to an envelope protein of dengue virus, wherein the antibody iscross-reactive with DENV serotype 1 envelope protein, DENV serotype 2envelope protein, DENV serotype 3 envelope protein, and DENV serotype 4envelope protein to protect a subject from dengue virus infection. Inone embodiment, the envelope protein is domain III of the envelopeprotein. The disclosure provides for a composition comprising a humananti-dengue virus antibody that binds to an envelope protein of denguevirus, wherein the antibody is cross-reactive with DENV serotype 1envelope protein, DENV serotype 2 envelope protein, DENV serotype 3envelope protein, and DENV serotype 4 envelope protein to protect asubject from dengue virus infection. In one embodiment, the envelopeprotein is domain III of the envelope protein.

The disclosure provides for the use of a human anti-dengue virusantibody that binds to an envelope protein of dengue virus, wherein theantibody is cross-reactive with DENV serotype 1 envelope protein, DENVserotype 2 envelope protein, DENV serotype 3 envelope protein, and DENVserotype 4 envelope protein to protect a subject from dengue virusinfection. In one embodiment, the envelope protein is domain III of theenvelope protein. The disclosure provides for the use of a compositioncomprising a human anti-dengue virus antibody that binds to an envelopeprotein of dengue virus, wherein the antibody is cross-reactive withDENV serotype 1 envelope protein, DENV serotype 2 envelope protein, DENVserotype 3 envelope protein, and DENV serotype 4 envelope protein toprotect a subject from dengue virus infection. In one embodiment, theenvelope protein is domain III of the envelope protein.

The disclosure also provides for use of a human anti-dengue virusantibody that binds to an envelope protein of dengue virus, wherein theantibody is cross-reactive with DENV serotype 1 envelope protein, DENVserotype 2 envelope protein, DENV serotype 3 envelope protein, and DENVserotype 4 envelope protein in the manufacture of a medicament for theprotection of a subject from dengue virus infection. In one embodiment,the envelope protein is domain III of the envelope protein. Thedisclosure also provides for use of a composition comprising a humananti-dengue virus antibody that binds to an envelope protein of denguevirus, wherein the antibody is cross-reactive with DENV serotype 1envelope protein, DENV serotype 2 envelope protein, DENV serotype 3envelope protein, and DENV serotype 4 envelope protein in themanufacture of a medicament for the protection of a subject from denguevirus infection. In one embodiment, the envelope protein is domain IIIof the envelope protein.

The disclosure also provides a composition comprising a proteincomprising an epitope that elicits production of an antibody withidentifying characteristics of a human anti-dengue virus antibodyselected from the group consisting of antibody m366, antibody m366.6,antibody m360.6, and antibody m360. Such a composition can also includea carrier. One embodiment is a pharmaceutical composition comprising aprotein comprising an epitope that elicits production of an antibodywith identifying characteristics of a human anti-dengue virus antibodyselected from the group consisting of antibody m366, antibody m366.6,antibody m360.6, and antibody m360. Such a pharmaceutical compositioncan also include a pharmaceutically acceptable carrier. Carriers andpharmaceutically acceptable carriers are known to those skilled in theart. Examples include, but are not limited to, aqueous solutions, suchas a buffer, such as a physiologically compatible buffer. Such acomposition can also include an adjuvant. Adjuvants to select are wellknown to those skilled in the art. A composition, such as apharmaceutical composition, of the embodiments is convenientlyformulated in a form suitable for administration to a subject.Techniques to formulate such compositions are known to those skilled inthe art.

The disclosure provides a method to protect a subject from dengue virusinfection, wherein the method comprises administering to the subject aprotein comprising an epitope that elicits production of an antibodywith identifying characteristics of a human anti-dengue virus antibodyselected from the group consisting of antibody m366, antibody m366.6,antibody m360.6, and antibody m360. In one embodiment, a pharmaceuticalcomposition comprising such an antibody is administered. As used herein,the ability of an epitope-containing protein of the embodiments toprotect a subject from dengue virus infection refers to the ability ofthe antibody to treat, prevent, or ameliorate the symptoms of denguevirus infection. In one embodiment, an epitope-containing protein of theembodiments prevents dengue virus infection in a subject. In oneembodiment, an epitope-containing protein of the embodiments treatsdengue virus infection in a subject. In one embodiment, anepitope-containing protein of the embodiments prevents symptoms ofdengue virus infection in a subject. In one embodiment, anepitope-containing protein of the embodiments treats symptoms of denguevirus infection in a subject. In one embodiment, an epitope-containingprotein of the embodiments prevents symptoms of dengue virus infectionfrom worsening in a subject. In one embodiment, an epitope-containingprotein of the embodiments ameliorates symptoms of dengue virusinfection in a subject. In one embodiment, an epitope-containing proteinof the embodiments reduces symptoms of dengue virus infection in asubject. Symptoms of dengue virus infection are known to those skilledin the art. Examples of symptoms include, but are not limited to, highfever, severe headache, severe eye pain, joint pain, muscle pain, skinrash, bleeding (e.g., nose or gum bleed, petechiae, easy bruising), lowwhite cell count, low platelet count, blood plasma leakage, low bloodpressure due to dengue shock syndrome, and dengue hemorrhagic fever. Insome cases, high fever is combined with one or more additional symptoms.Methods to identify and measure such symptoms are known to those skilledin the art.

As described herein, a subject is any animal that is susceptible todengue virus infection. Subjects include humans and non-human primates.In one embodiment, a subject is a human. In one embodiment, a subject isa non-human primate.

An epitope-containing protein of the embodiments can be administered ina variety of ways, such as by oral, intranasal, intraocular,conjunctival, intravenous, intraperitoneal, intramuscular, subcutaneous,intradermal, transdermal, topical, aerosol, and rectal administrationroutes.

The dose of an epitope-containing protein disclosed herein to beadministered to a subject to be effective (i.e., to protect a subjectfrom dengue virus infection) will depend on the subject's condition,manner of administration, and judgment of the prescribing physician. Oneskilled in the art can determine the dose as well as a dosing regimenthat defines the number and scheduling of doses required. Often a singledose is sufficient; however, the dose can be repeated if desirable.

An epitope-containing protein of the disclosure can be administeredalone or in combination with one or more other epitope-containingproteins. Examples of other proteins include, but are limited to, otherepitope-containing proteins as well as immunogens against otherdiseases. Examples of other anti-dengue epitope-containing proteinsinclude one or more epitope-containing proteins of the disclosure. Oneembodiment is a cocktail of epitope-containing proteins, such as acocktail of epitope-containing proteins of the disclosure. Anepitope-containing protein of the disclosure can also be administered incombination with one or more other therapeutic agents.

The disclosure provides a treatment for dengue virus infectioncomprising a protein comprising an epitope that elicits production of anantibody with identifying characteristics of a human anti-dengue virusantibody selected from the group consisting of antibody m366, antibodym366.6, antibody m360.6, and antibody m360, and a pharmaceuticallyacceptable carrier. The disclosure provides a preventative compositionagainst dengue virus infection comprising a protein comprising anepitope that elicits production of an antibody with identifyingcharacteristics of a human anti-dengue virus antibody selected from thegroup consisting of antibody m366, antibody m366.6, antibody m360.6, andantibody m360, and a pharmaceutically acceptable carrier.

The disclosure provides for a protein comprising an epitope that elicitsproduction of an antibody with identifying characteristics of a humananti-dengue virus antibody selected from the group consisting ofantibody m366, antibody m366.6, antibody m360.6, and antibody m360 toprotect a subject from dengue virus infection. The disclosure providesfor a composition comprising a protein comprising an epitope thatelicits production of an antibody with identifying characteristics of ahuman anti-dengue virus antibody selected from the group consisting ofantibody m366, antibody m366.6, antibody m360.6, and antibody m360 toprotect a subject from dengue virus infection.

The disclosure provides for the use of a protein comprising an epitopethat elicits production of an antibody with identifying characteristicsof a human anti-dengue virus antibody selected from the group consistingof antibody m366, antibody m366.6, antibody m360.6, and antibody m360 toprotect a subject from dengue virus infection. The disclosure providesfor the use of a composition comprising a protein comprising an epitopethat elicits production of an antibody with identifying characteristicsof a human anti-dengue virus antibody selected from the group consistingof antibody m366, antibody m366.6, antibody m360.6, and antibody m360 toprotect a subject from dengue virus infection.

The disclosure also provides for use of a protein comprising an epitopethat elicits production of an antibody with identifying characteristicsof a human anti-dengue virus antibody selected from the group consistingof antibody m366, antibody m366.6, antibody m360.6, and antibody m360 inthe manufacture of a medicament for the protection of a subject fromdengue virus infection. The disclosure also provides for use of acomposition comprising a protein comprising an epitope that elicitsproduction of an antibody with identifying characteristics of a humananti-dengue virus antibody selected from the group consisting ofantibody m366, antibody m366.6, antibody m360.6, and antibody m360 inthe manufacture of a medicament for the protection of a subject fromdengue virus infection.

Diagnosis of DENV Infection

The disclosure provides a method to diagnose dengue virus infectioncomprising use of an antibody of the embodiments or anepitope-containing protein of the embodiments. Such a method can be anin vitro, in vivo, or ex vivo diagnostic method. One embodiment is amethod to diagnose dengue virus infection in a subject comprising: (a)exposing a human anti-dengue virus antibody that binds to an envelopeprotein of dengue virus, wherein the antibody is cross-reactive withDENV serotype 1 envelope protein, DENV serotype 2 envelope protein, DENVserotype 3 envelope protein, and DENV serotype 4 envelope protein, to asample collected from the subject; and (b) detecting complex formationbetween the antibody and an epitope in the sample, wherein complexformation indicates that the subject is infected with dengue virus. Inone embodiment, the envelope protein is domain III of the envelopeprotein.

One embodiment is a method to diagnose dengue virus infection in asubject comprising: (a) exposing a human anti-dengue virus antibody thatbinds to an envelope protein of dengue virus, wherein the antibody iscross-reactive with DENV serotype 1 envelope protein, DENV serotype 2envelope protein, DENV serotype 3 envelope protein, and DENV serotype 4envelope protein, to the subject; and (b) detecting complex formationbetween the antibody and an epitope in the subject, wherein complexformation indicates that the subject is infected with dengue virus. Inone embodiment, the envelope protein is domain III of the envelopeprotein.

One embodiment is a method to diagnose dengue virus infection in asubject comprising: (a) exposing a protein comprising an epitope thatelicits production of an antibody with identifying characteristics of ahuman anti-dengue virus antibody selected from the group consisting ofantibody m366, antibody m366.6, antibody m360.6, and antibody m360 to asample collected from the subject; and (b) detecting complex formationbetween the epitope and an antibody in the sample, wherein complexformation indicates that the subject is infected with dengue virus.

One embodiment is a method to diagnose dengue virus infection in asubject comprising: (a) exposing a protein comprising an epitope thatelicits production of an antibody with identifying characteristics of ahuman anti-dengue virus antibody selected from the group consisting ofantibody m366, antibody m366.6, antibody m360.6, and antibody m360 tothe subject; and (b) detecting complex formation between the epitope andan antibody in the subject, wherein complex formation indicates that thesubject is infected with dengue virus. Methods to use such antibodiesand epitope-containing proteins of the embodiments to diagnose denguevirus infection are known to those skilled in the art.

The disclosure provides a diagnostic kit comprising a human anti-denguevirus antibody that binds to an envelope protein of dengue virus,wherein the antibody is cross-reactive with DENV serotype 1 envelopeprotein, DENV serotype 2 envelope protein, DENV serotype 3 envelopeprotein, and DENV serotype 4 envelope protein. In one embodiment, theenvelope protein is domain III of the envelope protein.

The disclosure also provides a diagnostic kit comprising proteincomprising an epitope that elicits production of an antibody withidentifying characteristics of a human anti-dengue virus antibodyselected from the group consisting of antibody m366, antibody m366.6,antibody m360.6, and antibody m360. Methods to make and use suchdiagnostic kits are known to those skilled in the art.

The following is a listing of the SEQ ID NOs disclosed in theapplication. It is to be appreciated that since nucleic acid sequencingtechnology is not entirely error-free, the nucleic acid sequences andamino acid sequences presented herein represent, respectively, apparentnucleic acid sequences of nucleic acid molecules of the embodiments andapparent amino acid sequences of proteins of the embodiments.

SEQ ID NO: Species Description 1 Synthetic Nucleic acid sequenceencoding human anti- dengue virus antibody m366 (VH-Linker-VL) 2Synthetic Human anti-dengue virus antibody m366 (VH-Linker-VL) 3Synthetic Nucleic acid sequence encoding m366 VH 4 Synthetic Humananti-dengue virus antibody m366 VH 5 Synthetic Nucleic acid sequenceencoding m366 VL 6 Synthetic Human anti-dengue virus antibody m366 VL 7Synthetic Nucleic acid sequence encoding m366 CDR-H1 8 Synthetic Humananti-dengue virus antibody m366 CDR-H1 9 Synthetic Nucleic acid sequenceencoding m366 CDR-H2 10 Synthetic Human anti-dengue virus antibody m366CDR-H2 11 Synthetic Nucleic acid sequence encoding m366 CDR-H3 12Synthetic Human anti-dengue virus antibody m366 CDR-H3 13 SyntheticNucleic acid sequence encoding m366 CDR-L1 14 Synthetic Humananti-dengue virus antibody m366 CDR-L1 15 Synthetic Nucleic acidsequence encoding m366 CDR-L2 16 Synthetic Human anti-dengue virusantibody m366 CDR-L2 17 Synthetic Nucleic acid sequence encoding m366CDR-L3 18 Synthetic Human anti-dengue virus antibody m366 CDR-L3 19Synthetic Nucleic acid sequence encoding m366 linker 20 Synthetic m366linker (Translation of SEQ ID NO: 19) 21 Synthetic Nucleic acid sequenceencoding human anti- dengue virus antibody m366.6 (VH-Linker-VL) 22Synthetic Human anti-dengue virus antibody m366.6 (VH-Linker-VL) 23Synthetic Nucleic acid sequence encoding m366.6 VH 24 Synthetic Humananti-dengue virus antibody m366.6 VH 25 Synthetic Nucleic acid sequenceencoding m366.6 VL 26 Synthetic Human anti-dengue virus antibody m366.6VL 27 Synthetic Nucleic acid sequence encoding m366.6 CDR-H1 28Synthetic Human anti-dengue virus antibody m366.6 CDR-H1 29 SyntheticNucleic acid sequence encoding m366.6 CDR-H2 30 Synthetic Humananti-dengue virus antibody m366.6 CDR-H2 31 Synthetic Nucleic acidsequence encoding m366.6 CDR-H3 32 Synthetic Human anti-dengue virusantibody m366.6 CDR-H3 33 Synthetic Nucleic acid sequence encodingm366.6 CDR-L1 34 Synthetic Human anti-dengue virus antibody m366.6CDR-L1 35 Synthetic Nucleic acid sequence encoding m366.6 CDR-L2 36Synthetic Human anti-dengue virus antibody m366.6 CDR-L2 37 SyntheticNucleic acid sequence encoding m366.6 CDR-L3 38 Synthetic Humananti-dengue virus antibody m366.6 CDR-L3 39 Synthetic Nucleic acidsequence encoding m366.6 linker 40 Synthetic m366.6 linker (Translationof SEQ ID NO: 39) 41 Synthetic Nucleic acid sequence encoding humananti- dengue virus antibody m360.6 (VH-Linker-VL) 42 Synthetic Humananti-dengue virus antibody m360.6 (VH-Linker-VL) 43 Synthetic Nucleicacid sequence encoding m360.6 VH 44 Synthetic Human anti-dengue virusantibody m360.6 VH 45 Synthetic Nucleic acid sequence encoding m360.6 VL46 Synthetic Human anti-dengue virus antibody m360.6 VL 47 SyntheticNucleic acid sequence encoding m360.6 CDR-H1 48 Synthetic Humananti-dengue virus antibody m360.6 CDR-H1 49 Synthetic Nucleic acidsequence encoding m360.6 CDR-H2 50 Synthetic Human anti-dengue virusantibody m360.6 CDR-H2 51 Synthetic Nucleic acid sequence encodingm360.6 CDR-H3 52 Synthetic Human anti-dengue virus antibody m360.6CDR-H3 53 Synthetic Nucleic acid sequence encoding m360.6 CDR-L1 54Synthetic Human anti-dengue virus antibody m360.6 CDR-L1 55 SyntheticNucleic acid sequence encoding m360.6 CDR-L2 56 Synthetic Humananti-dengue virus antibody m360.6 CDR-L2 57 Synthetic Nucleic acidsequence encoding m360.6 CDR-L3 58 Synthetic Human anti-dengue virusantibody m360.6 CDR-L3 59 Synthetic Nucleic acid sequence encodingm360.6 linker 60 Synthetic m360.6 linker (Translation of SEQ ID NO: 59)61 Synthetic Nucleic acid sequence encoding human anti- dengue virusantibody m360 (VH-Linker-VL) 62 Synthetic Human anti-dengue virusantibody m360 (VH-Linker-VL) 63 Synthetic Nucleic acid sequence encodingm360 VH 64 Synthetic Human anti-dengue virus antibody m360 VH 65Synthetic Nucleic acid sequence encoding m360 VL 66 Synthetic Humananti-dengue virus antibody m360 VL 67 Synthetic Nucleic acid sequenceencoding m360 CDR-H1 68 Synthetic Human anti-dengue virus antibody m360CDR-H1 69 Synthetic Nucleic acid sequence encoding m360 CDR-H2 70Synthetic Human anti-dengue virus antibody m360 CDR-H2 71 SyntheticNucleic acid sequence encoding m360 CDR-H3 72 Synthetic Humananti-dengue virus antibody m360 CDR-H3 73 Synthetic Nucleic acidsequence encoding m360 CDR-L1 74 Synthetic Human anti-dengue virusantibody m360 CDR-L1 75 Synthetic Nucleic acid sequence encoding m360CDR-L2 76 Synthetic Human anti-dengue virus antibody m360 CDR-L2 77Synthetic Nucleic acid sequence encoding m360 CDR-L3 78 Synthetic Humananti-dengue virus antibody m360 CDR-L3 79 Synthetic Nucleic acidsequence encoding m360 linker 80 Synthetic m360 linker (Translation ofSEQ ID NO: 79) 81 Synthetic DENV envelope protein, serotype 2(Swiss-Prot: P14338.1: DENV envelope serotype 2, modified to match SEQID NO: 82 between amino acid 291 and amino acid 395) 82 Synthetic DENVenvelope protein domain III.2 (serotype 2) 83 Synthetic Mouseanti-dengue virus antibody 9F12 VH 84 Synthetic Mouse anti-dengue virusantibody 9F12 VL 85 Synthetic Mouse anti-dengue virus antibody 9F12CDR-H1 86 Synthetic Mouse anti-dengue virus antibody 9F12 CDR-H2 87Synthetic Mouse anti-dengue virus antibody 9F12 CDR-H3 88 SyntheticMouse anti-dengue virus antibody 9F12 CDR-L1 89 Synthetic Mouseanti-dengue virus antibody 9F12 CDR-L2 90 Synthetic Mouse anti-denguevirus antibody 9F12 CDR-L3 91 Synthetic Nucleic acid encoding a humanIgG1 Fc with N- and C-terminal linkers 92 Synthetic Human IgG1 Fc withlinkers 93 Synthetic Nucleic acid sequence encoding mutated human IgG1Fc with N- and C-terminal linkers 94 Synthetic Mutated human IgG1 Fcwith linkers 95 Synthetic Nucleic acid sequence encoding bispecificm3666 96 Synthetic Bispecific anti-dengue virus antibody m3666 97Synthetic DENV 1, domain III consensus sequence 98 Synthetic DENV 2,domain III consensus sequence 99 Synthetic DENV 3, domain III consensussequence 100 Synthetic DENV 4, domain III consensus sequence 101Synthetic Nucleic acid sequence encoding human IgG1 Fc 102 SyntheticHuman IgG1 Fc 103 Synthetic Nucleic acid sequence encoding mutated humanIgG1 Fc 104 Synthetic Mutated human IgG1 Fc 105 Synthetic N-terminallinker of SEQ ID NO: 92 and SEQ ID NO: 94 106 Synthetic C-terminallinker of SEQ ID NO: 92 and SEQ ID NO: 94

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, and temperature is in degrees Celsius. Standardabbreviations are used.

Example 1 Materials and Methods

This Example provides the materials and methods for the other examplesof this section.

A. Yeast Display Naïve Human Antibody Library, Domain III Genes,Antibodies, Biotinylation Kit, Cells.

A large yeast display naïve single chain variable fragment (scFv) humanantibody library was constructed using a collection of human antibodygene repertoires, including the genes used for the construction of aphage display Fab library (Zhu Z et al., 2009, Methods Mol Biol 525,129-142, xv; and unpublished data from Zhu Z and Dimitrov DS laboratory,National Cancer Institute, Frederick, Md.).

All of the nucleic acid molecules encoding envelope domain III proteins(also referred to herein as ED3) were synthesized at Genscript, Inc.(Piscataway, N.J.). These nucleic acid molecules also containedappropriate restriction enzyme recognition sites at the flankingsequences for cloning purposes. Mouse monoclonal anti-c-Myc antibody waspurchased from Roche (Pleasanton, Calif.). PE-conjugated streptavidinand Alexa-488 conjugated goat anti-mouse antibody were purchased fromInvitrogen (Carlsbad, Calif.). Protein G columns were purchased from GEhealthcare (Waukesha, Wis.). Avi-tag specific biotinylation kits werepurchased from Avidity (Aurora, Colo.). Yeast plasmid extraction kitswere purchased from Zymo Research (Irvine, Calif.). 293 free styleprotein expression kits were purchased from Invitrogen. An AutoMACSSystem was purchased from Miltenyi Biotec (Cologne, Germany).

B. Mutant Design, DENV Envelope Domain III-Fc Fusion Protein Expression,Purification and Biotinylation

Envelope domain III fusion proteins from each of the four DENV serotypeswere produced as described below. These DIII.1, DIII.2, DIII.3, orDIII.4 fusion proteins were fused either to an Avi-tagged human IgG1 Fcor to a c-Myc-tagged human IgG1 Fc to enable efficient purification. Amutant DIII.3 protein fused to an Avi-tagged human IgG1 Fc was alsoproduced.

Nucleic acid molecules encoding dengue envelope domain III proteins fromthe 4 serotypes were synthesized at Genscript, using consensus geneinformation for each of the serotypes; the consensus sequences areprovided in FIG. 2. Nucleic acid molecules encoding DENV envelopeDIII.1, DIII.2, DIII.3, or DIII.4, each with a nucleic acid moleculeencoding an Avi- or c-Myc-tagged human IgG Fc domain fused at the 3′ endof the DNA encoding the respective DIII protein, were each cloned into apSecTag vector (Invitrogen), to produce recombinant molecules encodingeach respective tagged DIII. These recombinant molecules were namedpSecTag:DIII.1-Fc-Avi, pSecTag:DIII.1-Fc-cMyc, pSecTag:DIII.2-Fc-Avi,pSecTag:DIII.2-Fc-cMyc, pSecTag:DIII.3-Fc-Avi, pSecTag:DIII.3-Fc-cMyc,pSecTag:DIII.4-Fc-Avi, and pSecTag:DIII.4-Fc-cMyc.

A nucleic acid molecule encoding a mutant DENV envelope domain 111.3protein with a K310E substitution in the conserved neutralizing epitopeof domain 111.3 (serotype 3) was produced by introducing a pointmutation at the appropriate site of a nucleic acid molecule encodingDIII.3 using overlapping PCR. The 3′ end of the resultant nucleic acidmolecule was joined to a nucleic acid molecule encoding a Fc-Avi-tag,and cloned into pSecTag to produce a recombinant molecule encoding themutant DIII.3, named pSecTag:DIII.3(K310E)-Fc-Avi.

Each of the recombinant molecules was transfected into 293 free stylecells to produce the respective DIII proteins following themanufacturer's protocol. The respective proteins were purified using aprotein G column. Biotinylation of the purified DENV envelope DIII.1-Fc,DIII.2-Fc, DIII.3-Fc, and DIII.4-Fc proteins with Avi-tags was performedusing the kit from Avidity following the manufacturer's instructions.The mutant DENV envelope DIII.3-Fc protein was not biotinylated.

C. MACS Sorting Downsize of the Initial Yeast Display Human AntibodyLibrary

Biotinylated DENV envelope domain III.3-Fc was used as the target forthree rounds of sorting to downsize the initial yeast display naïvehuman antibody library. Approximately 10¹⁰ cells from the initial naïveantibody library and 10 μg of biotinylated domain III.3-Fc wereincubated in 50 ml PBSA (phosphate-buffered saline containing 1% bovineserum albumin) at room temperature (RT) for 2 hr with rotation; one mgof unrelated antibody (m102.4 IgG1) with the same Fc was also mixed inthe mixture to eliminate any Fc-specific antibodies from binding to theFc domain of the biotinylated domain III.3-Fc. The mixture ofbiotinylated DIII.3-Fc binding to displayed antibody on cells from thelibrary was washed three times with PBSA and incubated at RT with 100 μlof streptavidin conjugated microbeads from Miltenyi Biotec. Theresultant mixture was washed once with PBSA at RT and loaded onto theAutoMACS system for the first round of sorting. The sorted cells wereamplified in SDCAA media (20 g dextrose, 6.7 g Difco yeast nitrogen basew/o amino acids, 5 g Bacto casamino acids, 5.4 g Na₂HPO₄ and 8.56 gNaH₂PO₄. H₂O in 1 liter water) at 30° C. and 250 rpm for 24 hr. Theculture was then induced in SGCAA media (20 g galactose, 20 g raffinose,1 g dextrose, 6.7 g Difco yeast nitrogen base w/o amino acids, 5 g Bactocasamino acids, 5.4 g Na₂HPO₄ and 8.56 g NaH₂PO₄. H₂O in 1 liter water)at 20° C. and 250 rpm for 16-18 hr.

The same amounts of antigen, control antibody m102.4 IgG1, andincubation volume were used for the next two rounds of sorting. The cellnumbers used for these two rounds of sorting were set at 100 times thesize of the sorted pool from the previous round of sorting to keep thediversity of each sorted pool.

D. Competitive Library Panning and Monoclonal Yeast Clone Screening byFACS

The downsized library was further sorted against the biotinylated domainIII.3-Fc fusion protein on a FACSAria II cell sorter using theunbiotinylated domain III.3-Fc mutant bearing the point mutation (K310E)in the neutralizing epitope as a competitor. Briefly, 5×10⁸ cells wereincubated with 1 μg/ml of biotinylated domain 111.3 Fc fusion protein,10 μg/ml of unbiotinylated domain 111.3 mutant Fc fusion protein, and 2μg/ml of mouse anti-c-Myc antibody in 5 ml of PBSA with rotation for 2hr at RT followed by three washes with PBSA. A 1:100 dilutedPE-conjugated streptavidin and Alexa-488 conjugated goat anti-mouseantibody were then mixed with the cells and incubated at RT for 30 minin the dark, followed by another two washes with PBSA. The stained cellswere loaded on the cell sorter for sorting. The gate was set to collectthe population with the brightest PE/Alexa signal. The collected cellswere amplified in SDCAA media and induced in SGCAA medium as describedabove. The induced cells were sorted one more time following the sameprocess, and the sorted cells were analyzed on FACS to confirm thespecificity of the sorting. The sorted pool, containing only the cellsthat could not be competed by the mutant, was plated on a SDCAA platefor single yeast clone analysis. Random yeast clones from the plate wereinoculated into liquid SDCAA medium for amplification, and induced at20° C. in 2 ml SGCAA medium for 16 hr. Induced monoclonal yeast cellswere stained as described as above without the mutant competitor andanalyzed on FACS to select the positive binders.

E. Expression and Purification of scFv-Fc-Avi Proteins

Plasmids were extracted from the identified yeast clones using yeastplasmid extraction kits (Zymo Research), following the manufacturer'sinstructions. Extracted plasmids were transformed into 10 G chemicalcompetent E. coli (Lucigen , Middleton, Wis.) for further amplification;plasmids extracted from the bacteria were used for DNA sequencing toobtain the nucleic acid sequences encoding the positive binderantibodies. The scFv-encoding inserts of the unique clones were digestedwith SfiI and ligated into modified pSecTag bearing the same set of SfiIsites and Fc-Avi tag for soluble expression. These constructs weretransfected into 293 free style cells for expression following themanufacturer's protocol. After 72 hr of growth, the scFv-Fc fusionproteins were purified from the cell culture medium using protein Gcolumns.

F. ELISA Binding Assay

The purified scFv-Fc proteins were each diluted into PBS atconcentration 2 μg/ml; 50 μl of the diluted proteins were coated in a96-well plate at 4° C. overnight. The c-Myc-tagged DIII proteins fromall four serotypes were each serially diluted in 3% milk-PBS and addedto the antibody-coated wells for 1 hr after blocking with 3% milk-PBS atRT. After washing, a 1:2000 diluted HRP conjugated anti-c-Myc antibodywas added for 1 hr at RT. After washing, 3, 3, 5,5′-Tetramethylbenzidine (TMB) substrate was added, and the O.D. was readat 450 nm.

G. Mutant Library Construction Through Error-Prone and DNA Shuffling PCR

The scFv D6 mutant library was generated by introducing random pointmutations into the DNA encoding scFv D6 through error-prone polymerasechain reaction (PCR), using a GeneMorph II kit (Stratagene, AgilentTechnologies, Santa Clara, Calif.) following the manufacturer's protocolwith minor modifications. To further diversify the mutation profile, 3μM of each of the nucleotide analogues 8-oxo-deoxyguanosine triphosphateand 2′-deoxy-p-nucleoside-5′-triphosphate was mixed in the PCR reactionmixture. For the second and third cycle library constructions, an extrastep of DNA shuffling PCR was inserted into the regular PCR cycles tocombine the beneficial mutations obtained from previous maturationprocess; see, e.g., Zhao H et al, 1998, Nature Biotechnology 16,258-261, for a description of DNA shuffling PCR. The DNA shuffling PCRstep was performed as following: 20 cycles of denaturation at 94° C. for15 seconds followed by annealing/extension at 68 ° C. for 1 second onthe Biorad MyCycler (BioRad, Hercules, Calif.).

H. Affinity Determination by Surface Plasmon Resonance

Binding affinities of human anti-DENV scFv m366 to the DENV envelope (E)domain III proteins of the four serotypes were analyzed by surfaceplasmon resonance technology using a Biacore X100 instrument (GEhealthcare). The scFv m366 was covalently immobilized onto a sensor chip(CM5) using carbodiimide coupling chemistry. A control reference surfacewas prepared for nonspecific binding and refractive index changes. Foranalysis of the kinetics of interactions, varying concentrations ofantigens were injected at a flow rate of 30 μl/min using running buffercontaining 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% SurfactantP-20 (pH 7.4). The association and dissociation phase data were fittedsimultaneously to a 1:1 Langmuir global model, using the nonlinear dataanalysis program BlAevaluation 3.2. All the experiments were done at 25°C.

I. Plaque Reduction Assay

Serially diluted antibodies in 100 μl L-15 medium (Sigma-Aldrich, St.Louis, Mo.) containing 1% FCS were mixed with 100 μl virus and incubatedin a 24-well plate at 37° C. for 1 hr. An 0.5 ml aliquot of PS clone Dcells (4×10⁵/ml) (Sriburia R et al., 2001, J Virological Methods 92, 71)were added and allowed to adhere at 37° C. for 2 hr. Then 0.5 ml ofoverlay medium (3% carboxymethylcellulose in L15 with 5% FCS) was addedand followed by incubation at 37° C. for 5 to 6 days. Cells were stainedwith naphthalene black.

J. Epitope Mapping of Isolated Antibodies Through Domain III MutantLibrary Sorting

Random point mutations were introduced into the dengue virus envelopeprotein domain 111.2 coding region (serotype 2) through error-prone PCRusing the GeneMorph II mutagenesis kit from Stratagene following themanufacturer's protocol. A repertoire of gel purified gene mutants wasre-amplified using YDRDF and YDRDR in regular PCR conditions to add theflanking sequences for in vivo recombination through the Gap repairingprocess (Zhao Q et al., 2011, Mol Cancer Ther 10, 1677-1685). SfiIdigested and gel purified yeast display vector pYD7 (Zhao Q et al.,ibid.) was mixed with the mutant gene repertoire and transformed intoelectroporation-competent yeast cells prepared as described (Perez B etal., 2010, Protein Eng Des 23, 155-159). Yeast display domain 111.2mutant library amplification and induction were performed as describedin the Examples herein. The induced mutant library (5×10⁸ cells) wasincubated with 1 μg/ml biotinylated ScFv D6-Fc and 2 μg/ml mouseanti-c-Myc antibody at RT for 2 hr, followed by three washes, andincubation with a mixture of PE-conjugated streptavidin and Alexa-488conjugated goat anti-mouse antibody as described in the Examples herein.Stained cells were loaded onto the cell sorter; cells that lackedbinding to the antigen even though it was still expressed on the yeastcell surface, as demonstrated by Alexa Fluor 488 staining only, weresorted. The sorting process was repeated once under the same conditions.Sorted cells after the second round were amplified; plasmids wereextracted from the pool using yeast plasmid extraction kits (ZymoResearch), amplified in 10 G E. coli, and sequenced.

K. Computational Docking of Complex Between Env-DIII and scFv Antibodym366

Homology modeling of the variable regions (or domains) of heavy (V_(H))and light (V_(L)) chains for the scFv antibody m366 was carried outusing the SWISS-MODEL workspace by selecting the closest templatestructures (PDB codes: 3QOS for heavy chain and 2DD8 for light chain),with sequence similarities of 92% and 87% respectively. The V_(H)-V_(L)orientation of the scFv m366 structure was assigned to be similar to oneof the templates (PDB code: 2DD8) that showed minimal steric clash forcreating the final scFv m366 model. The crystal structure of theEnv-DIII protein of DENV serotype 2 (PDB code: 2R29) was used fordocking with the modeled scFv antibody m366. Docking of scFv m366 to theDENV Env-DIII.2 was performed by accessing the ZDOCK server at theUniversity of Massachusetts Medical School's ZLab (Zhiping's Lab) website. The ZDOCK server uses a fast Fourier transform (FFT)-basedrigid-body protein docking algorithm with scoring functions combiningpairwise shape complementarity, desolvation and electrostatic energies.Based on the escape mutants that led to the loss of epitopes andavailable crystal structure of DENV Env-DIII, residues 307, 309, 310,311, 327, 361 and 383, on the surface of Env-DIII were selected aspotential contacting residues for docking constraints. Similarly, one ortwo residues from each of the CDR-H1, CDR-H3 and CDR-L3 loops werechosen at the docking interface. The CDR-H1 and CDR-H3 loops haddominant hydrophobic residues, whereas CDR-L1 had a germline mutation,and they all had high antigen-contacting propensities. Results from thetop 2000 ZDOCK predictions were filtered using the user-defined residuesand a 6-angstrom distance cutoff. Three predicted complexes were onlykept as all residues selected come together at the interface and werefurther examined by PDBePISA (Protein Interfaces, Surfaces andAssemblies). PyMOL (available at the PyMOL website, Schrodinger, NewYork) was used for the analysis of docked model and graphicalillustration.

Example 2 Identification of a Human Anti-Dengue Virus Cross-ReactiveAntibody That Binds to Dengue Virus Envelope Domain III Proteins Fromall 4 Serotypes

This Example describes the identification a human anti-dengue virusantibody of the embodiments that is cross-reactive with DENV serotype 1envelope protein, DENV serotype 2 envelope protein, DENV serotype 3envelope protein, and DENV serotype 4 envelope protein.

AutoMACS is an ideal platform to quickly sort large numbers of yeastcells and downsize the initial naive library to make it feasible for theFACS-based cell sorter. Biotinylated DENV envelope domain III.3-Fc (alsoreferred to as DENV Env-DIII.3-Fc, domain III.3-Fc, or DIII.3-Fc) wasused for three rounds of sorting of a yeast display naive antibodylibrary with size at 5×10⁹ on AutoMACS. An excessive amount of anunrelated m102.4 IgG1 with the same Fc fragment as domain III.3-Fcfusion protein was mixed in the library to deplete the potential Fcspecific binders. Three rounds of sorting on AutoMACS dramaticallyenriched the domain III.3-Fc binders. The binding of the entire doublepositive population to the domain 111.3 Fc fusion protein could becompeted by a protein that contained only DENV envelope domain 111.3(i.e., without Fc), indicating the binding of the enriched pool wasdomain III specific, and depletion of potential anti-Fc bindingantibodies (Fc binders) using m102.4 IgG1 was efficient.

The downsized pool showed specific binding to the biotinylated DENVenvelope domain III.3-Fc with the target antigen at 1 μg/ml. When 10μg/ml of the unbiotinylated domain III.3-Fc mutant bearing the pointmutation (K310E) and 1 μg/ml of the biotinylated wild type domainIII.3-Fc were mixed with the yeast pool, the FACS profile obtained fromthe cell sorter showed the pool divided into several populations, aseach was impacted by the mutant competition to a different extent. Thefirst cell population represented those cells that could not bind to theunbiotinylated domain III.3-Fc mutant but still bound well to thebiotinylated wild type domain III.3-Fc. The second cell populationrepresented those cells that bound well to epitopes shared by both thewild type and mutant domain 111.3. The third cell population, cells thatshowed no competition by the mutant, were sorted out and plated on aSDCAA plate for single clone screening.

FACS analysis was performed to screen the plated random clones from thesorted yeast pool, which could not be competed by the designed mutantK310E. Plasmids were extracted from the positive yeast clones andsequenced. Two unique clones, designated as D6 and D7, were identified,and nucleic acid molecules encoding their scFv inserts were eachintroduced into a pSecTag vector encoding the Avi-tagged human IgG1 Fcdomain such that the Avi-tagged human IgG1 Fc domain of the resultantprotein was at the carboxyl terminus (C terminus) of the scFv fusionprotein. The resultant proteins were called scFv D6-Fc-Avi and scFvD7-Fc-Avi.

Human anti-dengue virus antibody scFv D6-Fc-Avi was submitted to anELISA binding assay as described herein. FIG. 3 shows that the proteinencoded by clone D6 (i.e., scFv D6) bound dengue virus envelope domainIII proteins from all 4 serotypes.

Example 3 Affinity Maturation Through Random Mutagenesis andQuantitatively Yeast Library Sorting

This Example demonstrates that the cross-reactive binding activity ofhuman anti-dengue virus antibody D6 was maintained through the affinitymaturation that yielded human anti-dengue virus antibody m366.

After three cycles of mutagenesis and selection of a library encodingscFv D6, one clone was identified from monoclonal yeast display antibodyscreening of the enriched pool after the final round of sorting,designated as scFv m366. The scFv m366 gene was cloned into a pSecTagvector encoding human IgG1 Fc for scFv m366-Fc fusion proteinexpression. Biacore analysis showed that the cross-reactive bindingactivities of scFv m366 to DENV Env domain Ills from all four DENVserotypes was preserved after the affinity maturation process.

The data are presented in Table 1.

TABLE 1 Binding affinity of scFv m366 to DENV envelope domain IIIs from4 serotypes measured by Biacore. DENV Env Protein Domains k_(a) (1/Ms)k_(d) (1/s) K_(D) (nM) DIII serotype 1 8.601E+5 0.02058 6.136 DIIIserotype 2 8.689E+5 0.02039 2.508 DIII serotype 3 1.863E+5 2.039E−41.095 DIII serotype 4 2.659E+5 0.01032 12.38

The nucleic acid sequence encoding human anti-dengue virus antibody m366is provided in nucleic acid sequence SEQ ID NO:1. The amino acid ofantibody m366 is provided in amino acid sequence SEQ ID NO:2. Thenucleic acid sequences encoding the V_(H) and V_(L) chains of antibodym366 are provided in SEQ ID NO:3 and SEQ ID NO:5, respectively. Theamino acid sequences of the V_(H) and V_(L) chains of antibody m366 areprovided in SEQ ID NO:4 and SEQ ID NO:6, respectively. A comparison ofthe amino acid sequences of the V_(H) and V_(L) chains of humananti-dengue virus antibody m366 and mouse anti-dengue virus antibody9F12, a mouse monoclonal antibody that neutralizes all four dengue virusserotypes (see, e.g., Rajamanonmani R et al., ibid.) indicates that thehuman and mouse antibodies show less than 50 percent sequence identity,even in their respective CDRs. The amino acid sequence alignment isshown in FIG. 8.

Example 4 Neutralization of Dengue Virus Isolates From all 4 Serotypesby scFv m366-Fc

This Example demonstrates the ability of a human anti-dengue virusantibody of the embodiments to neutralize isolates from DENV serotype 1,DENV serotype 2, DENV serotype 3, and DENV serotype 4.

A plaque reduction assay was performed to evaluate the neutralizationactivity of scFv m366. FIG. 4 provides data demonstrating that scFvm366-Fc potently neutralized the infections of dengue viruses fromserotypes 2 and 3. Table 2 provides data indicating that, at 25 μg/ml,scFv m366-Fc fusion proteins neutralized the isolates from 4 allserotypes.

TABLE 2 Summary of the neutralization activity of scFv antibody m366-Fc.Antibodies (25 μg/ml) D6 D7 D3.12 M366 DENV-1 − − − + DENV-2 − − − +DENV-3 − − − + DENV-4 − − − + JEV − − − −

Example 5 Epitope Mapping Through Yeast Display Domain III Escape MutantIdentification and Sequence Analysis

This Example uses epitope mapping analysis to localize the DENV envelopedomain III epitope recognized by a human anti-dengue virus antibody ofthe embodiments. It provides epitope mapping data that shows theposition of amino acids mutations in mutant DENV envelope domain IIIproteins that can no longer bind to a human anti-dengue virus antibodyof the embodiments. Such mutated residues are thought to indicate thelocation of the epitope recognized by the antibody.

A serotype 2 derived DENV envelope domain III consensus gene was used asa template for error-prone PCR. The resulting mutant gene repertoire wascloned into a yeast display vector through gap repairing to construct ayeast display DENV envelope domain 111.2 mutant library of 2×10⁸ cells.Two rounds of sorting of yeast cells that expressed mutant forms of DENVenvelope DIII.2 protein on the cell surface that lacked binding to thescFv D6-Fc fusion protein were performed. Yeast cells from the secondround of sorting were collected; plasmids were extracted from this pooland amplified in 10G E. coli. Plasmids were extracted from 48 singlecolonies, and sequenced.

Sequence analysis showed that all of the clones encoded completeproteins in the correct reading frame, which is in agreement with thefact that all of the sorted clones expressed mutant DIII.2 proteins onthe yeast cell surface. Furthermore, all the clones had at least onemutation encoding a mutated protein, with up to 5 mutations in someclones. Any mutants bearing mutations that occurred in the two cysteineor in solvent inaccessible residues were excluded from sequenceanalysis. Amino acid sequences from 35 binding escape mutants werealigned with the consensus protein sequence of DENV envelope domain IIIserotype 2. Mutation frequency at each position was plotted against theamino acid residue position number. Similarly, 193 unique envelopedomain III amino acid sequences derived from naturally isolated serotype2 dengue viruses from GenBank were also aligned with the consensussequence. The superimposed profiles of the two sets of sequences in FIG.5 demonstrate that the majority of mutations leading to binding escapemutants are located in regions of Env-DIII.2 that are typically wellconserved in nature. Without being bound by theory, this result mightexplain the broad cross-reactivity of m366 to naturally isolated dengueviruses.

Example 6 Epitope Analysis Using the Docked Model of DENV Env-DIII-scFvAntibody m366 Complex

This Example provides additional information regarding the localizationof the DENV envelope domain III epitope recognized by a humananti-dengue virus antibody of the embodiments.

Computational docking of the complex between dengue virus Env-DIII andscFv antibody m366 (DENV Env-DIII-scFv antibody m366 complex) wasperformed using the ZDOCK method. Three docked complexes were selectedthat contained the key residues identified from the experimental epitopemapping approach described in the Examples herein. One of the top scoreddocked models exhibited minimum clashes with appropriate proteininterface parameters and was used to demonstrate the location of thepotential epitopes and their interactions with antibody m366. Withoutbeing bound by theory, it is believed that such identification can shedlight on the molecular mechanisms of the broadly cross-reactiveneutralization of human anti-dengue virus antibodies of the embodiments.FIG. 6 shows the docking model of the Env-DIII-scFv antibody m366complex in which the epitopes are highlighted. The docking modelrevealed a different orientation of antibody binding to Env-DIII in thecomplex structure of FIG. 6 compared to that of Env-DIII with Fab-1A1D-2previously determined (Lok S M et al., 2008, ibid.). The m366 epitopeidentified herein is comprised of three structurally proximal regions:residues 305-311; 325, 327 and 361; and 383 and 385 at the C-terminalend. One of the key residues, K310, mutation of which of affects epitopeformation, contacts the CDR-L1 of m366; CDR-L1 has a germline mutation.In the Env-DIII-Fab-1A1D-2 complex crystal structure, the residue K310contacts the CDR-H1. Hydrophobic residues, Ile and Trp, of CDR-H3contact the center part of the epitope and other loops H1, H2, L2 and L3are also involved in the binding. The surface area of the interfacebetween Env-DIII and scFv antibody m366 is 716 A², typical ofantibody-antigen interactions. There are six hydrogen bonds likely toform and no salt bridges at the interface. However, the DENVEnv-DIII-scFv antibody m366 complex crystal structure would ultimatelyclarify the precise molecular interactions. Interestingly, proteininterface similarity analysis of 1476422 interfaces showed two relevanthits, one is crystal structure of an antibody complexed with anthraxprotective antigen domain 4 and the other is the recent structure ofinfluenza A neutralizing antibody in complex with human H3 influenzahemagglutinin (Corti D et al., 2011, Science 333, 850-856).

Example 7 Summary and Conclusions

These Examples describe the identification of a human antibody m366,from a naïve human antibody library, that binds with high (i.e.,nanomolar) affinity to envelope domain III from all four DENV serotypesand neutralizes each of those serotypes. There are two majorimplications from this finding: First is that m366 is a potentialcandidate therapeutic that could be further developed in preclinical andclinical settings. Second is that the epitope of m366 could be used as acandidate vaccine immunogen capable of eliciting m366 and/or m366-likeantibodies.

As of August 2011, twenty-nine monoclonal antibodies have been approvedfor clinical use in the United States or Europe as well as six Fc fusionproteins (Dimitrov, submitted for publication). Only one of theseantibodies, Synagis, is against an infectious disease (caused by RSV),and it is used for prevention, not for therapy (Marasco WA et al.,ibid.). Previous studies have identified a candidate therapeutic humanmonoclonal antibody, m102.4, which potently neutralized both Hendra andNipah viruses although they differ with respect to their amino acidsequences; the sequence differences are about the same as between anytwo DENV serotypes (see, for example, Bossart K N et al., 2009, PLoSPathog. 5, e1000642; Zhu Z et al., 2008, J Infect Dis. 197, 846-853; ZhuZ et al., 2006, J Virol. 80, 891-899). Monoclonal antibody m102.4 wassuccessful as a candidate therapeutic mAb in a monkey disease modelwhere it cured animals treated three days after challenge withinfectious virus, while all control monkeys died; M102.4 was alsoadministered to three humans without side effects (Bossart et al.,submitted for publication). Antibody m102.4 was produced by goodmanufacturing practices (GMP) in Australia, and is in stock to be usedin future outbreaks.

Human anti-dengue antibody m366 was selected from the same yeast displaylibrary as m102.4, and was subjected to affinity maturation light chainshuffling similarly to the maturation of m102.4. The only differencesare that (a) m366 was selected by yeast display in a scFv format, whilem102.4 was selected by yeast display in an Fab format, (b) there wereadditional rounds of selection from mutant libraries for m366, and (c)to increase the probability for success, a domain III (D3) mutantcorresponding to a non-replicating virus was used to deplete the libraryfrom putative non-neutralizing antibodies.

Without being bound by theory, it is believed that these differencescould further increase the potency and breadth of neutralization bym366. Because m102.4 has been so successful and is likely to getapproval for clinical use by FDA based on the positive data from twoanimal models and lack of side effects, a similarly identified antibody,but with different specificity, could be a promising candidatetherapeutic. In progress are experiments in two animal models and with apanel of primary isolates that will provide further information for thepotential therapeutic utility of m366.

A combination of computational structural modeling and sequence analysisof mutants has been used to approximately localize the m366 epitope. Theepitope appears to overlap to some extent with epitopes previouslyexplored as targets for cross-reactive murine mAbs. This furtherindicates that the m366 epitope could be an important component ofvaccine immunogens intended to elicit cross-reactive neutralizingantibodies. In progress are experiments to crystallize the complex ofscFv m366 with DENV envelope domain III that would allow precisedetermination of the m366 epitope.

Example 8 Identification of Human Anti-Dengue Virus Cross-ReactiveAntibody m360

This Example describes the identification a human anti-dengue virusantibody that is cross-reactive to dengue virus envelope domain IIIproteins from three serotypes. Human anti-dengue virus antibody scFvD7-Fc-Avi, the production of which was described in Example 2, wassubmitted to an ELISA binding assay as described herein. FIG. 9 showsthat the protein encoded by clone D7 (i.e., scFv D7) bound dengue virusenvelope domain III proteins from DENV serotype 1, DENV serotype 2, andDENV serotype 3.

After three cycles of mutagenesis and selection of a library encodingscFv D7, one clone was identified from monoclonal yeast display antibodyscreening of the enriched pool after the final round of sorting,designated as scFv m360. The scFv m360 gene was cloned into a pSecTagvector encoding human IgG1 Fc for scFv m360-Fc fusion proteinexpression. Biacore analysis showed that the cross-reactive bindingactivities of scFv m360 to DENV Env domain Ills from three of the fourDENV serotypes was preserved after the affinity maturation process. Thedata are presented in Table 3.

TABLE 3 Binding affinity of scFv m360 to DENV envelope domain IIIsmeasured by Biacore DIII Serotype 1 DIII Serotype 2 DIII Serotype 3 DIIISerotype 4 K_(d) (M) K_(d) (M) K_(d) (M) K_(d) (M) 5.84E−9 5.13E−91.16E−10 8.28E−8

The nucleic acid sequence encoding human anti-dengue virus antibody m360is provided in nucleic acid sequence SEQ ID NO:61. The amino acid ofantibody m360 is provided in amino acid sequence SEQ ID NO:62. Thenucleic acid sequences encoding the V_(H) and V_(L) chains of antibodym360 are provided in SEQ ID NO:63 and SEQ ID NO:65, respectively. Theamino acid sequences of the V_(H) and V_(L) chains of antibody m360 areprovided in SEQ ID NO:64 and SEQ ID NO:66, respectively.

Example 9 Affinity Maturation of Additional Human Anti-Dengue VirusCross-Reactive Antibodies

This Example demonstrates the ability to affinity mature humananti-dengue virus cross-reactive antibodies m360 and m366 to obtainantibodies m360.6 and m366.6, respectively, with enhancedcharacteristics compared to m360 and m366.

Human anti-dengue virus cross-reactive antibodies m360 and m366 wereeach affinity matured as described in the Examples herein. For example,a library encoding m360 was mutated, submitted to one round ofMACS-based sorting, followed by three rounds of mutagenesis andselection by FACS to specifically isolate the pool of binders with thehighest binding activity; typically the sorting gate was set to sort out0.5%. 0.2% and 0.1% of the pool for rounds 1, 2 and 3 respectively. Thenthe final yeast pool-derived plasmid was used as template forerror-prone and DNA shuffling PCR (as described in the Examples herein)to construct the next mutant library for another cycle of mutagenesisand selection. A total three additional rounds of mutagenesis andselection by FACS were performed, and a single clone was identifiedafter the final round of sorting, designated as antibody m360.6.Antibody m366.6 was affinity matured from antibody m366 in a similarmanner. Biacore analysis showed that both scFv m360.6 and scFv m366.6bound to DENV Env domain Ills from all four DENV serotypes, as indicatedin Table 4.

TABLE 4 Binding affinities of scFv m360.6 and scFv m366.6 to DENVenvelope domain IIIs from 4 serotypes measured by Biacore. DIII Sero-DIII Sero- DIII Sero- DIII Sero- type 1 type 2 type 3 type 4 AntibodyK_(d) (nM) K_(d) (nM) K_(d) (nM) K_(d) (nM) m360.6 0.31 0.24 0.0023 33m366.6 0.44 0.29 0.27 0.75

The nucleic acid sequence encoding human anti-dengue virus antibodym360.6 is provided in nucleic acid sequence SEQ ID NO:41. The amino acidof antibody m360.6 is provided in amino acid sequence SEQ ID NO:42. Thenucleic acid sequences encoding the V_(H) and V_(L) chains of antibodym360.6 are provided in SEQ ID NO:43 and SEQ ID NO:45, respectively. Theamino acid sequences of the V_(H) and V_(L) chains of antibody m360.6are provided in SEQ ID NO:44 and SEQ ID NO:46, respectively. The nucleicacid sequence encoding human anti-dengue virus antibody m366.6 isprovided in nucleic acid sequence SEQ ID NO:21. The amino acid ofantibody m366.6 is provided in amino acid sequence SEQ ID NO:22. Thenucleic acid sequences encoding the V_(H) and V_(L) chains of antibodym366.6 are provided in SEQ ID NO:23 and SEQ ID NO:25, respectively. Theamino acid sequences of the V_(H) and V_(L) chains of antibody m366.6are provided in SEQ ID NO:24 and SEQ ID NO:26, respectively.

Example 10 Production of a Human Dengue Virus Bispecific Antibody, andComparison of Neutralization Titers of Human Dengue Virus Antibodies ofthe Embodiments

This Example demonstrates the production of a human dengue virusbispecific antibody, denoted m3666, comprising human dengue virusantibodies m360.6 and m366.6. This Example also demonstrates that humandengue virus antibodies m360.6, m366.6 and m3666 were each able toneutralize all four DENV serotypes, i.e., DENV serotype 1, DENV serotype2, DENV serotype 3, and DENV serotype 4.

Human dengue virus bispecific antibody m3666 was produced by cloning thenucleic acids encoding the scFv of m360.6 at the N terminus and m366.6at the C-terminus of a mutated human IgG1 Fc into mammalian expressionvector pSectag from Invitrogen. The mutant Fc (having amino acidsequence SEQ ID NO:94, encoded by nucleic acid sequence SEQ ID NO:93)was constructed by deleting the two leucines near the N-terminus of CH2of the non-mutated Fc (having amino acid sequence SEQ ID NO:92, encodedby nucleic acid sequence SEQ ID NO:91). SEQ ID NO:92 and SEQ ID NO:94contain, respectively, the non-mutated IgG1 Fc (SEQ ID NO:102) and themutated IgG1 Fc (SEQ ID NO:104), each flanked on the N-terminus by the7-amino acid linker GQAGQGP (SEQ ID NO:105) and on the C-terminus by the8-amino acid linker AAAGGGGS (SEQ ID NO:106). The bispecific antibodywas expressed and purified from 293 freestyle cells following the vendorprovided protocol.

The abilities of human dengue virus antibodies scFv-Fc m360.6, scFv-Fcm366.6 and bispecific m3666 to neutralize all four DENV serotypes wasdetermined by using a reporter system using pseudo-infectious DENVreporter virus particles (DENV RVP assay) as described by Mattia K etal., 2011, PLoS ONE 6 (11): e27252. Table 5 provides DENV neutralization(IC₅₀) data for the antibodies m360.6, m366.6 and m3666. Neutralizationdata for mouse monoclonal antibody 4G2 (available from ATCC CAT# HB-112,ATCC, Manassas, Va.) are also shown for comparison.

TABLE 5 DENV neutralization (IC₅₀) data for antibodies m360.6, m366.6,and m3666 measured by a DENV RVP assay. DIII Sero- DIII Sero- DIII Sero-DIII Sero- type 1 type 2 type 3 type 4 Antibody (μg/ml) (μg/ml) (μg/ml)(μg/ml) scFv-Fc 12 4.2 1.5 21 m360.6 scFv-Fc 22 2.4 0.85 0.36 m366.6m3666 0.80 0.22 0.07 0.01 4G2 2.0 2.0 1.8 0.30

Example 11 Enhancement of Infection by Human Anti-Dengue VirusAntibodies of the Embodiments

This Example demonstrates that human anti-dengue virus antibodies of theembodiments do not enhance infection by Dengue virus.

Viral enhancement assays were conducted against all four serotypes ofDENV (DENV-1 (WestPac), DENV-2 (S16803), DENV-3 (CH53489) and DENV-4(TVP360) to measure the potentially pathogenic activity of humananti-dengue virus antibody m366. All enhancements were run in replicatesin order to ensure accuracy. DENV RVPs (replication-incompetent virusparticles) were pre-incubated with an equal volume of serially dilutedantibody (25 μg/ml to 0.00127 μg/ml; all dilutions are pre-dilution) inRPMI complete for 1 hour at room temperature with slow agitation.Following incubation, human erythroleukemic Fc Rlla-bearing K562 cellswere added to each well at a density of 40,000 cells per well followedby incubation at 37° C. in 50% CO2 for 72 hours. Cells were subsequentlylysed and analyzed for luminescent reporter expression. Raw infectiondata expressed as luminescence was plotted versus log 10 of the antibodydilution, as shown in FIG. 11. mAb X is a negative control, and mAb Y isantibody m366. A dose response curve was applied for curve fitting todetermine the titer of antibody that achieved the highest level ofinfection. No infection level was determined by using a no antibodycontrol. The Figure demonstrates that human anti-dengue virus antibodym366 does not significantly enhance Dengue virus infection.

Human anti-dengue virus antibody m366.6, human anti-dengue virusantibody 360.6, and human anti-dengue virus bispecific antibody m3666were also evaluated and shown not to significantly enhance Dengue virusinfection.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims.

1-145. (canceled)
 146. A human anti-dengue virus antibody that binds todomain III of an envelope protein of dengue virus, wherein the antibodyis cross-reactive with domain III of dengue virus (DENV) serotype 1envelope protein, domain III of DENV serotype 2 envelope protein, domainIII of DENV serotype 3 envelope protein, and domain III of DENV serotype4 envelope protein, and wherein: (a) the antibody comprises acomplementarity determining region (CDR) having an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,and combinations thereof; or (b) the antibody binds an epitope bound bythe antibody comprising a complementarity determining region (CDR)having an amino acid sequence selected from the group consisting ofamino acid sequences SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, and combinations thereof.
 147. Thehuman anti-dengue virus antibody of claim 146, wherein the antibodycomprises a CDR-H3 having amino acid sequence SEQ ID NO:32, or theantibody binds an epitope bound by the antibody comprising a CDR-H3having amino acid sequence SEQ ID NO:32.
 148. The human anti-denguevirus antibody of claim 146, wherein the antibody neutralizes DENVserotype 1, DENV serotype 2, DENV serotype 3, and DENV serotype 4 ordoes not enhance Dengue virus infection or a combination thereof. 149.The human anti-dengue virus antibody of claim 146, wherein the antibodycomprises a scFv.
 150. The human anti-dengue virus antibody of claim146, wherein: (a) the antibody comprises a CDR-H1 having amino acidsequence SEQ ID NO:28, a CDR-H2 having amino acid sequence SEQ ID NO:30,a CDR-H3 having amino acid sequence SEQ ID NO:32, a CDR-L1 having aminoacid sequence SEQ ID NO:34, a CDR-L2 having amino acid sequence SEQ IDNO:36, and a CDR-L3 having amino acid sequence SEQ ID NO:38; or (b) theantibody binds an epitope bound by an antibody comprising a CDR-H1having amino acid sequence SEQ ID NO:28, a CDR-H2 having amino acidsequence SEQ ID NO:30, a CDR-H3 having amino acid sequence SEQ ID NO:32,a CDR-L1 having amino acid sequence SEQ ID NO:34, a CDR-L2 having aminoacid sequence SEQ ID NO:36, and a CDR-L3 having amino acid sequence SEQID NO:38.
 151. The human anti-dengue virus antibody of claim 146,wherein the antibody comprises an amino acid sequence that is at leastabout 90 percent identical to an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NO:24, SEQ ID NO:26, andSEQ ID NO:22.
 152. The human anti-dengue virus antibody of claim 146,wherein the antibody comprises an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NO:24, SEQ ID NO:26, andSEQ ID NO:22.
 153. The human anti-dengue virus antibody of claim 146,wherein the antibody comprises a V_(H) chain comprising an amino acidsequence that is at least about 90 percent identical to amino acidsequence SEQ ID NO:24 and a VL chain comprising an amino acid sequencethat is at least about 90 percent identical to amino acid sequence SEQID NO:26.
 154. The human anti-dengue virus antibody of claim 146,wherein the antibody comprises an antibody comprising a V_(H) chaincomprising amino acid sequence SEQ ID NO:24 and a VL chain comprisingamino acid sequence SEQ ID NO:26.
 155. The human anti-dengue virusantibody of claim 146, wherein the antibody comprises a first amino acidsequence that is at least about 90 percent identical to amino acidsequence SEQ ID NO:24 and a second amino acid sequence that is at leastabout 90 percent identical to amino acid sequence SEQ ID NO:26, whereinthe first amino acid sequence and second amino acid sequence are joinedby a peptide linker.
 156. The human anti-dengue virus antibody of claim146, wherein the antibody comprises amino acid sequence SEQ ID NO:24 andamino acid sequence SEQ ID NO:26, wherein amino acid sequence SEQ IDNO:24 and amino acid sequence SEQ ID NO:26 are joined by a peptidelinker.
 157. The human anti-dengue virus antibody of claim 146, whereinthe antibody comprises amino acid sequence SEQ ID NO:22.
 158. A nucleicacid molecule encoding a human anti-dengue virus antibody of claim 146.159. A recombinant cell comprising the nucleic acid molecule of claim158.
 160. A pharmaceutical composition comprising a human anti-denguevirus antibody of claim 146 and a pharmaceutically acceptable carrier.161. A method to protect a subject from dengue virus infection, themethod comprising administering to the subject a human anti-dengue virusantibody of claim
 146. 162. A method to produce a human anti-denguevirus antibody of claim 146, the method comprising: (a) culturing arecombinant cell encoding the antibody; and (b) recovering the antibody.163. A diagnostic kit comprising the human anti-dengue virus antibody ofclaim
 146. 164. A method to diagnose dengue virus infection in a subjectcomprising: (a) exposing a human anti-dengue virus antibody of claim 146to the subject or to a sample collected from the subject; and (b)detecting complex formation between the antibody and an epitope in thesubject or in the sample, wherein complex formation indicates that thesubject is infected with dengue virus.
 165. The human anti-dengue virusantibody of claim 146, wherein the antibody is not isolated from a humansubject.